Interferon-gamma-binding molecules for treating septic shock, cachexia, immune diseases and skin disorders

ABSTRACT

The present invention concerns molecules which bind and neutralize the cytokine interferon-gamma. More specifically, the present invention relates to sheep-derived antibodies and engineered antibody constructs, such as humanized single-chain Fv fragments, chimeric antibodies, diabodies, triabodies, tetravalent antibodies, peptabodies and hexabodies which can be used to treat diseases wherein interferon-gamma activity is pathogenic. Examples of such diseases are: septic shock, cachexia, multiple sclerosis and psoriasis.

This application is a divisional of co-pending application Ser. No.10/071,485, filed Feb. 7, 2002, which was a divisional of applicationSer. No. 09/485,737, filed Feb. 14, 2000, now U.S. Pat. No. 6,350,860,which was a section 371 national stage filing of PCT/EP98/05165, FiledAug. 14, 1998 (published in English on Feb. 25, 1999 As WO 99/09055) andclaiming priority to EP 97870122.5 filed Aug. 18, 1997; and EP98870139.7 filed Jun. 18, 1998, each of which are incorporated herein byreference.

FIELD OF THE INVENTION

The present invention concerns molecules which bind and neutralize thecytokine interferon-gamma. More specifically, the present inventionrelates to sheep-derived antibodies and engineered antibody constructs,such as humanized single-chain Fv fragments, chimeric antibodies,diabodies, triabodies, tetravalent antibodies and peptabodies which canbe used to treat diseases wherein interferon-gamma activity ispathogenic. Examples of such diseases are: septic shock, cachexia,multiple sclerosis and psoriasis.

BACKGROUND OF THE INVENTION

Interferon-gamma (IFNγ) is a member of the interferon family ofimmunomodulatory proteins and is produced by activated T helper type-1cells (Th1 cells) and natural killer cells (NK cells). Apart from itspotent antiviral activity, IFNγ is known to be involved in a variety ofimmune functions (for a review, see Billiau, 1996) and inflammatoryresponses. Indeed, IFNγ is the primary inducer of the expression of themajor histocompatibility complex (MHC) class-II molecules (Steinman etal., 1980) by macrophages and other cell types and stimulates theproduction of inflammatory mediators such as tumor necrosis factor-alpha(TNFα), interleukin-1 (IL-1) and nitric oxide (NO) (Lorsbach et al.,1993). In this respect, IFNγ is shown to be important in themacrophage-mediated defence to various bacterial pathogens. Furthermore,IFNγ is also shown to be a potent inducer of the expression of adhesionmolecules, such as the intercellular adhesion molecule-1 (ICAM-1, Dustinet al., 1988), and of important costimulators such as the B7 moleculeson professional antigen presenting cells (Freedman et al., 1991).Moreover, IFNγ induces macrophages to become tumoricidal (Pace et al.,1983) and provokes Ig isotype switching (Snapper and Paul, 1987).

The anti-viral, tumoricidal, inflammatory- and immunomodulatory activityof IFNγ clearly has beneficial effects in a number of clinicalconditions. However, there are a number of clinical situations in whichIFNγ-activity has deleterious effects. These include cancer cachexia(Denz et al., 1993; Iwagaki et al., 1995), septic shock (Doherty et al.,1992), skin disorders such as psoriasis and bullous dermatoses (Van denOord et al., 1995), allograft rejection (Landolfo et al., 1985;Gorczynski, 1995), chronic inflammations such as ulcerative colitis andCrohn's disease (WO 94/14467 to Ashkenazi & Ward), and autoimmunediseases such as multiple sclerosis (M S, Panitch et al., 1986),experimental lupus (Ozmen et al., 1995), arthritis (Jacob et al., 1989;Boissier et al., 1995) and autoimmune encephalomyelitis (Waisman et al.,1996).

Cachexia is a phenomenon often seen in cancer patients and is associatedwith losses of lean body mass, and altered carbohydrate and lipidmetabolism. This so called ‘chronic wasting syndrome’ is often theimmediate cause of death. In recent years, interest has focused on therole of proinflammatory cytokines in cancer related cachexia. Currentdata support the concept that cachexia is linked to the presence ofcertain cytokines among which IFNγ seems to play a central role. Denz etal. (1993) reported that increased neopterin and decreased tryptophanconcentrations—which are closely related to IFNγ-activity—are detectedin cachectic patients suffering from hematological disorders. Neopterinis synthesized and secreted by monocytes/macrophages upon stimulation byIFNγ from activated T cells. Tryptophan is an indispensable amino acidwhich can be catabolized by indoleamine 2,3-dioxygenase, an enzymeinduced by IFN's, and which absence initiates mechanisms responsible forcachexia (Brown et al., 1991). The correlation between high neopterinlevels, decreased tryptophan levels and weight loss was confirmed byIwagaki et al. (1995). In experimental models, cancer-induced cachexiacan be altered by the administration of IFNγ neutralizing antibodies(Matthys et al., 1991; Langstein et al., 1991)

Septic shock is the result of a severe bacterial infection, and remainsa common cause of death among critically ill, hospitalized patientsdespite improvements in supportive care (Bone et al., 1992). Althoughseptic shock may be associated with gram-positive infections, attentionhas focused on the more common pathogenesis of gram-negative sepsis andthe toxic role of endotoxin (=lipopolysaccharide or LPS), a component ofthe outer membrane of gram-negative and some gram-positive bacteria.Many of the effects of LPS are mediated through the release of cytokinessuch as TNFα (Tracey, 1991), IL-1 (Wakabayashi et al., 1991) and IFNγ(Bucklin et al., 1994). Much of the evidence supporting the role ofthese cytokines as mediators of septic shock comes from lethalitystudies involving the blockade of individual cytokines, resulting inprotection of experimental animals from otherwise lethal doses ofendotoxin or gram-negative bacteria. One of the first events in septicshock is the activation of T cells by antigen presenting cells ontowhich bacterial superantigen is bound (Miethke et al., 1993). Uponactivation, for which co-stimulation of CD28 is essential (Saha et al.,1996), these T cells proliferate and produce a surge of proinflammatorycytokines such as IL-2, TNFα and IFNγ eventuating in the clinicalsyndrome. Also, it is hypothesized that LPS induces the expression ofthe α1/β1 integrin (VLA-1) heterodimer on activated monocytes which thendisplay an increased capacity to adhere to the endothelial basementmembrane. Similar effects can be induced by incubation of monocytes withIFNγ (Rubio et al., 1995). VLA-1 might also contribute to furthermonocyte activation and potentiation of the production ofmonocyte-derived pro-inflammatory cytokines during sepsis (Rubio et al.,1995). Although very promising results were obtained with fantibodiesneutralising TNFα in experimental animal models, clinical trials withanti-TNFα antibodies revealed only a slight reduction or even noreduction in mortality rate of patients with septic shock (Wherry etal., 1993; Reinhart et al., 1996). A -fusion protein containing theextracellular portion of the TNF receptor and the Fc portion of IgG1also did not affect mortality (Fisher et al., 1996). Pentoxifylline(PTX), a methyl xanthine derivative, is currently being tested for itseffect on the outcome of septic shock. PTX is known to lower the serumconcentrations of at least TNFα, IL-1 and IFNγ (Bienvenu et al., 1995;Zeni et al., 1996). Initial data reveal that PTX leads to an improvementof the clinical status of septic patients (Mandi et al., 1995). There isevidence that IFNγ is a mediator of lethality during sepsis. Antibodiesthat either neutralize IFNγ or block the IFNγ-receptor are protectingagainst lethality (Bucklin et al., 1994; Doherty et al., 1992). Asynergistic effect between IFNγ and TNFα has also been suggested(Doherty et al., 1992; Ozmen et al., 1994). Although not in itselflethal, IFNγ has been shown to be essential for the manifestation ofTNF-induced lethality in the generalized Shwartzman reaction (Ozmen etal., 1994).

Bullous, inflammatory and neoplastic dermatoses are a heterogenous groupof skin disorders during which IFNγ may play a pathogenic role. Bullousdermatoses encompass epidermolysis bullosa acquisita, bullous pemhigoid,dermatitis herpetiformes During, linear IgA disease, herpes gestationis,cicatricial pemhigoid, bullous systemic lupus erythematosis,epidermolysis bullosa junctionalis, epidermolysis bullosa dystrophicans,porphyria cutanea tarda and Lyell-Syndrome (Megahed, 1996). Alsoerythema exsudativum multiform major (Kreutzer et al., 1996),IgG-mediated subepidermal bullous dermatosis (Chan & Cooper, 1994),bullous lichen planus (Willsteed et al., 1991) and paraneoplasticbullous dermatosis (Pantaleeva, 1990) can be classified among thebullous dermatoses. A pathogenic role of IFNγ during bullous dermatoseshas been suggested by Van den Oord et al. (1995). The role of IFNγduring inflammatory and neoplastic dermatoses, compared to bullousdermatoses, has been more extensively investigated. Indeed, it has beendemonstrated that IFNγ is involved during the pathogenesis of verrucosis(Asadullah et al., 1997), eosinophilic pustular folliculitis (Teraki etal., 1996), cutaneous T cell lymphoma (Wood et al., 1994), granulomafaciale (Smoller & Bortz, 1993), Sweet's syndrome (Reuss-Borst et al.,1993), atopic eczema (Arenberger et al., 1991), follicular mucinosis(Meisnerr et al., 1991), lichen-planus and psoriasis (Vowels et al.,1994). One of the most extensively studied inflammatory dermatoses ispsoriasis. Psoriasis is a hyperproliferative skin disorder affectingapproximately 2% of the population. Evidence is accumulating that thedisease has a T-cell mediated autoimmune etiology. The role of T-cellsin psoriasis has been demonstrated by Gottlieb et al. (1995). The latterauthors suggested that, in most of the patients, clinical andhistopathological features of psoriasis are primarily linked to skininfiltration by IL-2 receptor-positive leukocytes. Disease improvementcan be induced by the administration of a fusion protein composed ofhuman interleukin-2 and fragments of diphteria toxin, which selectivelyblocks the growth of activated lymphocytes. Other effectiveanti-psoriatic, T-cell suppressing agents include the immunosuppressivedrugs cyclosporin and FK506 (Griffiths, 1986) and anti-CD4 monoclonalantibodies (Morel et al., 1992). More direct evidence for the role of Tcells in the induction of the complex tissue alterations seen inpsoriasis has been generated by Schön et al. (1997) using a model withscid/scid mice in which they transferred naive, minor histocompatibilitymismatched CD4⁺ T-cells, resulting in the development of a skin disorderthat resembles psoriasis. The autoimmune character of the disease hasbeen proposed by Valdimarsson et al. (1995) who stated that products ofactivated T-cells can induce keratinocytes of individuals with psoriaticpredisposition to express determinants that are recognized by T cellsspecific for epitopes on β-haemolytic streptococci. Several data suggestthat IFNγ may play a crucial role in the pathogenesis of psoriasis.IFNγ, produced by activated T cells would be involved in the recruitmentof lymphocytes (Nickoloff, 1988), in the induction of activation andadhesion molecules on epidermal keratinocytes (Dustin et al., 1988), aswell as in the abnormal keratinocyte proliferation (Barker et al.,1993). Not only enhanced levels of IFNγ has been detected in psoriaticepidermis (Kaneko et al., 1990), also de novo suprabasal expression ofIFNγ receptor in psoriasis has been demonstrated (Van den Oord et al.,1995).

Inflammatory bowel disease (IBD), which encompasses ulcerative colitisand Crohn's disease, is characterized by the appearance of lesions ofunknown aetiology in most parts of the gut. IBD is rather common, with aprevalence in the range of 70-170 in a population of 100,000. Thecurrent therapy of IBD involves the administration of anti-inflammatoryor immunosuppressive agents, which usually bring only partial results,and surgery. In view of the apparent shortcomings of the presenttreatment, Ashkenazi and Ward (WO 94/14467) suggested the usage of abispecific antibody construct targeting IFNγ and another molecule, suchas IL-1 and TNFα, to treat IBD. However, the exact role of IFNγ duringIBD is not well understood.

MS is a severely disabling progressive neurological disease of unknownaetiology, but probably involving autoimmune responses and resulting inthe appearance of focal areas of demyelinisation (Williams et al.,1994). MS affects 1 in 1000 persons in the USA and Europe, but due toimproved diagnosis that number is increasing. Onset of disease isusually around 30 years of age and, on average, patients are in need oftreatment for another 28 years. MS is among the most expensive chronicdiseases of western society, based on duration and intensity of care.However, diagnosis of exacerbations and early identification of onset ofexacerbations has improved greatly, allowing design of novel treatmentstrategies. Active multiple sclerosis lesions feature T-lymphocyte andmonocyte-macrophage accumulations at plaque margins where myelin isbeing destroyed. The inflammatory cells that invade the white matter andthe soluble mediators that they release are held primarily responsiblefor myelin breakdown. Population-based studies indicate that certainHLA-antigens occur with higher frequency in patients with MS (withpredominant MHC being the Dw2(DR2)DQ1.2 haplotype (Olerup et al., 1991).Similar associations of class I and class II haplotypes have also beendetected in other autoimmune disorders such as rheumatoid arthritis andinsulin dependent diabetes (Nepom, 1993). The lesions of MS arecomparable to those found in chronic relapsing experimental allergicencephalitis (EAE), an autoimmune disease that can be induced in animalsby immunization with e.g. whole myelin (Allen et al., 1993) or with themyelin/oligodendrocyte glycoprotein (Genain et al., 1995b). The lesionsassociated with EAE are similar in appearance as the ones occurring inMS and also contain inflammatory infiltrates of T-cells and macrophages(Genain, et al., 1995b). Furthermore, in adoptive transfer experiments,T cells sensitized to specific myelin antigens can transfer the diseasestate of EAE (Genain et al., 1995b; Waldburger et al., 1996). A fewyears ago, the American FDA approved the use of the immunosuppressivedrug interferonβ (trade name Betaseron) for treatment of chronicrelapsing MS. The effect of this drug—although modest—clearlydemonstrates the involvement of the cytokine network in thepathophysiology of MS. In the last few years, a large number of studieshave addressed the molecular mechanism by which Betaseron exerts itsbeneficial effects. Lately, it was shown that IFNβ dose-dependentlyinhibited T-cell proliferation, expression of IL-2 receptors andsecretion of IFNγ, TNFα and IL-13 (Rep et al., 1996). Furthermore, itwas demonstrated that IFNβ could specifically prevent the IFNγ-inducedup regulation of MHC class II antigens and adhesion molecules onantigen-presenting cells (Jiang et al., 1995) and human brainmicrovessel endothelial cells (Huynh et al., 1995).

One of the earliest events in MS is damage of the blood brain barrier(BBB) by activated, encephalitogenic T-cells (Tsukada et al., 1993). Themechanism by which these cells destruct locally the BBB, which is mainlyconstituted of endothelial cells, is not elucidated, but it is knownthat at the systemic level, local production of certain cytokines suchas IFNγ enhance the capability of lymphocytes to adhere to endothelialcells (Yu et al., 1985; Tsukada et al., 1993). Also, on choroid plexusepithelial cells of EAE animals, an increased expression of ICAM-1 andVCAM-1 (Steffen et al., 1994), for which LFA-1 and VLA-4 are the naturalligands on lymphocytes, has been observed. Mc Carron et al. (1993)reported that adhesion of MBP-specific T lymphocytes was significantlyup regulated when cerebral endothelial cells were treated with IL-1,TNFα or IFNγ. That the adhesion of encephalitogenic T-cells to theendothelium is an early and very important event in the onset of MS isshown by the finding that anti LFA-1 therapy can completely block theinduction of EAE (Gordon et al., 1995). Additional circumstantialevidence for a stimulatory role of IFNγ in the pathophysiology of MScomes from observations that disease exacerbations are induced by viralupper respiratory infections, known to stimulate the secretion of IFNγby type-2 helper T cells (Panitch, 1994). The proinflammatory role ofIFNγ in autoimmune disease is strengthened by an earlier finding thattreatment of MS patients with hIFNγ resulted in an aggravation of thesymptoms (Panitch et al., 1986). The role of IFNγ as proinflammatorycytokine in autoimmune disorders has been studied in severalexperimentally induced forms of autoimmunity. In experimental neuritis,induced by myelin or antigen-specific T cells in rat, IFNγ clearly actedas pro-inflammatory cytokine and administration of a monoclonal antibodyto IFNγ suppressed the disease (Hartung et al., 1990). In the case ofexperimental autoimmune thyroiditis (EAT) in mice, induced by theinjection of thyroglobulin, treatment of the animals with anti-IFNγ at 4weeks after induction of EAT proved to be beneficial, sincecharacteristic features of EAT such as the lymphocytic infiltrations ofthe thyroid glands and the serum levels of autoantibodies tothyroglobulin, were significantly reduced (Tang et al., 1993).

In the mouse EAE model for MS, where the disease can be induced byinjection of either spinal cord homogenate or myelin basic protein,elevated concentrations of several cytokines, including IFNγ wereobserved both in serum and in the lesions in the CNS (Willenborg et al.,1995). However, administration of anti-IFNγ at the initiation of thedisease, resulted in an exacerbation of the disease (Billiau et al.,1988; Duong et al., 1994; Willenborg et al., 1995). It must be noted,however, that in these experiments the effect of anti-IFNγ wasdetermined at the onset of acute EAE rather than at the time of chronicrelapse of the disease, which in fact is the only relevant situation forMS. Pathologically, typical acute EAE differs substantially from MS inthat prominent inflammation occurs in gray, white and meningealstructures, but demyelisation is scant or absent (Genain et al., 1995b).In order to explain the findings with anti-IFNγ antibodies, the authorssuggest a different action of IFNγ at the systemic level(anti-inflammatory action) compared to the local level (inflammatoryaction) (Billiau et al., 1988), or suggest an early role (within 24 hafter immunization) of IFNγ in disease resistance (Duong et al., 1994).Willenborg et al. (1995) conclude that the time of treatment plays acritical role on the outcome and suggest this to be the explanation forconflicting results in different autoimmune processes. Recently,Heremans et al. (1996) described facilitation of spontaneous relapses inchronic relapsing EAE in Biozzi ABH mice by administration of anti-IFNγduring the remission phase. The onset of relapses was delayed whenanimals were treated with IFNγ during the remission phase, results whichare in contradiction to the excacerbation seen in humans who weretreated with hIFNγ.

An experimental EAE model that more closely resembles the disease courseand symptomatology of MS in humans can be found in marmosets. Indeed, inthese animals a chronic relapsing-remitting form of EAE can be inducedwhich is characterized by an initial, acute phase with clinically mildneurological signs, followed by recovery. A late spontaneous relapseoccurs in these animals and chronic lesions resemble active plaques ofchronic MS (Massacesi et al., 1995). This unique model can efficientlybe employed to evaluate a prospective therapy for MS. In this model, acritical role for TNFα in demyelisation is suggested by the observationthat rolipram, a selective inhibitor of the type IV phosphodiesterase,suppressed TNFα secretion and demyelisation (Genain et al., 1995a;Sommer et al., 1995) when administered shortly after immunization, thusinterfering with acute EAE. The effect of anti-IFNγ on acute EAE or ondisease relapse has to our knowledge never been investigated inmarmoset.

Taken together, it is well established that there are a number ofclinical situations in which IFNγ-activity has deleterious effects.Consequently, several potential therapies to neutralize IFNγ-activityhave been proposed. Among the latter proposals are the use of: anti-IFNγantibodies (Ozmen et al., 1995; Bucklin et al., 1994), recombinantanti-IFNγ Fv fragments (EP 0528469 to Billiau & Froyen), bispecificmolecules (WO 94/14467 to Ashkenazi and Ward), drugs such aspentoxifylline (Bienvenu et al., 1995), synthetic polypeptides whichinhibit binding of IFNγ to its receptor (U.S. Pat. No. 5,451,658 toSeelig; U.S. Pat. No. 5,632,988 to Ingram et al.), Epstein-Barr virusderived proteins (U.S. Pat. No. 5,627,155 to Moore & Kastelein), solubleIFNγ receptors (EP 0393502 to Fountoulakis et al.; U.S. Pat. No.5,578,707 to Novick & Rubinstein) and oligonucleotides which bind toIFNγ (WO95/00529 to Coppola et al.). However, these compounds are facedwith problems such as suboptimal stability, affinity and clearancerates, lack of specificity, efficacy and tissue penetrance, toxic sideeffects and unwanted carrier effects. Indeed, the carrier effect ofantibodies can limit their efficiency to block the target cytokine. Forexample, Montero-Julian et al. (1995) showed that during treatment ofmyeloma patients with anti-IL-6, accumulation of IL-6 in the serum inthe form of monomeric immune complexes occurred, hereby stabilizing thecytokine. Furthermore, it has also been shown that the therapeuticefficacy of a cytokine can be prolonged by the formation ofcytokine/antibody complexes, since the efficacy of recombinant humanIL-2 treatment could be increased by prolonging its in vivo half-life bycomplexing with an anti-IL-2 antibody (Courtney et al., 1994). Thecarrier-effect of anti-cytokine antibodies can be overcome by theconstruction of monovalent scFv fragments, although their low MW(∀30.000) and the associated fast clearance rate, make them lesssuitable candidates for long-term treatment. However, the undesirablecarrier effect can be avoided by the formation of higher immunecomplexes, as such increasing the clearance of the cytokine-antibodycomplexes (Montero-Julian et al., 1995). The use of monoclonalantibodies for diagnostic or therapeutic purposes in vivo is, besidesthe carrier effect, also limited because of their nature (i.e. themajority are murine mAb's and administration of antibodies of mouseorigin inevitably results in a human anti-mouse antibody [HAMA]response), their suboptimal efficacy, stability and affinity and theirlarge molecular size. Proposed solutions to some of these problemsinvolve the use of F(ab′)2, F(ab) and scFv derivatives or of humanizedversions of the parent antibody, either by CDR grafting (Kettleboroughet al., 1991) or by resurfacing of the antibodies (Roguska et al.,1994). Another proposed solution is the development of several modifiedantibodies or antibody constructs by bioengineering or chemical methods.Indeed, some mAb's were made more effective by conjugatingchemotherapeutic drugs and other toxins to the antibodies (Ghetie andVitetta, 1994) or by developing bispecific and/or multivalent antibodyconstructs capable of simultaneously binding several -or two differentepitopes on the same- or different antigens. These antibody constructshave been produced using a variety of methods: a) antibodies ofdifferent specificities or univalent fragments of pepsin-treatedantibodies of different specificities have been chemically linked(Fanger et al., 1992); b) two hybridomas secreting antibodies ofdifferent specificity have been fused and the resulting bispecificantibodies from the mixture of antibodies were subsequently isolated; c)genitically engineered single chain antibodies have been used to producenon-covalently linked bispecific antibodies (e.g. diabodies (Holliger etal., 1993), minibodies (Kostelny et al., 1992) and tetravalentantibodies (Pack et al; 1995; WO 96/13583 to Pack) or covalently-linkedbispecific antibodies (e.g. chelating recombinant antibodies (Kranz etal., 1995), single chain antibodies fused to protein A or Streptavidin(Ito and Kurosawa, 1993; Kipriyanov et al., 1996) and bispecifictetravalent antibodies (EP 0517024 to Bosslet and Deeman). Recently,also trivalent antibody constructs, named triabodies (Kortt et al.,1997), and pentavalent constructs, named peptabodies (Terskikh et al.,1997), have been described. These constructs may have a higher avidityin comparison to bivalent constructs and may be useful for diagnostic ortherapeutic purposes in vivo.

However, and despite the fact that several potential therapies toneutralize IFNγ-activity have been proposed, no prior art existsregarding the production and existence of engineered antibodyconstructs, such as humanized single-chain Fv fragments, diabodies,triabodies, tetravalent antibodies, peptabodies and hexabodies, andruminant-derived antibodies such as sheep antibodies which overcome theabove-indicated problems and which can efficiently be used to treatdiseases wherein interferon-gamma activity is pathogenic.

SUMMARY OF THE INVENTION

It is clear from the prior art as cited above that problems such assuboptimal stability, affinity, clearance rate, specificity, efficacy,and an unwanted carrier effect and HAMA response hamper the successfulusage of several therapeutics which, potentially, could neutralize theactivity of IFNγ. Also suggested solutions to overcome some of theseproblems did not result in the development of effective products. Thus,unpredictable and unknown factors still appear to determine the successof these biologicals. Despite these unknown factors, the presentinventors have been able to design and develop useful constructs whicheffectively neutralize IFNγ-activity. Indeed, the constructs have all asurprisingly high affinity for IFNγ, they do not provoke a HAMA orrelated response, and they do not result in a carrier effect. Inaddition, some of the constructs pass the blood brain barrier, whereasothers have a very good clearance rate. Therefore, the present inventionaims at providing a molecule which binds and neutralizesinterferon-gamma and which is chosen from the group consisting of:

-   -   a scFv comprising the humanized variable domain of the        monoclonal antibody D9D 10    -   a chimeric antibody comprising the humanized variable domain of        the monoclonal antibody D9D10    -   a diabody comprising the humanized variable domain of the        monoclonal antibody D9D10    -   a multivalent antibody    -   a ruminant antibody.

The present invention further aims at providing a multivalent antibodychosen from the group consisting of triabodies, tetravalent antibodies,peptabodies and hexabodies.

The present invention also aims at providing a triabody, tetravalentantibody, peptabody and hexabody which comprise 3, 4, 5 and 6 variabledomains, respectively, of different anti-interferon-gamma antibodies.

The present invention further aims at providing a triabody as describedabove which comprises 3 identical variable domains of ananti-interferon-gamma antibody. A preferred variable domain used in thelatter constructs is derived from the mouse anti-interferon-gammaantibody D9D10 which is described by Sandvig et al. (1987) and Froyen etal. (1993) or from the sheep anti-interferon-gamma antibody described inthe present application. Therefore, the present invention aims atproviding a triabody as described above which comprises 3 identicalD9D10 scFv's, 3 identical humanized D9D10 scFv's, 3 identicalsheep-derived anti-interferon-gamma scFv's or 3 identical humanizedsheep-derived anti-interferon-gamma scFv's.

The present invention further aims at providing a tetravalent antibody(called MoTAb I) as described above which comprises 4 identical domainsof an anti-interferon-gamma antibody. More specifically, the presentinvention aims at providing a tetravalent antibody as described abovewhich comprises either 4 identical D9D10 scFv's or 4 identicalsheep-derived anti-interferon-gamma scFv's in the format of a homodimerof 2 identical molecules, each containing 2 D9D10 scFv's or 2 humanizedD9D10 scFv's or 2 sheep-derived anti-interferon-gamma scFv's or 2humanized sheep-derived anti-interferon-gamma scFv's, and a dimerizationdomain, or, a full-size humanized D9D10 antibody or sheep-derivedanti-interferon-gamma antibody to which 2 humanized D9D10 scFv's or 2humanized sheep-derived anti-interferon-gamma scFv's, respectively, areattached at the carboxyterminus (called MoTAb II) (see FIG. 1).

The present invention further aims at providing a peptabody and hexabodyas described above which comprise 5 and 6 identical variable domains ofan anti-interferon-gamma antibody, respectively. A preferred variabledomain used in the latter constructs is derived from the mouseanti-interferon-gamma antibody D9D10 which is described above or fromthe sheep anti-interferon-gamma antibody described in the presentapplication. Therefore, the present invention aims at providing apeptabody and hexabody as described above which comprises 5 or 6identical D9D10 scFv's, 5 or 6 identical humanized D9D10 scFv's, 5 or 6identical sheep-derived anti-interferon-gamma scFv's, or, 5 or 6identical humanized sheep-derived anti-interferon-gamma scFv's,respectively.

The present invention further aims at providing a molecule as describedabove, wherein said ruminant antibody is a sheep antibody.

The present invention also aims at providing a molecule as describedabove, wherein said sheep antibody is a monoclonal antibody.Furthermore, the present invention aims at providing a humanizedantibody, a single-chain fragment or any other fragment which is derivedfrom said monoclonal antibody and which has largely retained thespecificity of said monoclonal antibody.

Moreover, the present invention aims at providing methods for producingthe above-described molecules.

The present invention further aims at providing a pharmaceuticalcomposition comprising a molecule as described above, or a mixture ofsaid molecules, in a pharmaceutically acceptable excipient.

The present invention also aims at providing a molecule or a compositionas described above for use as a medicament.

Furthermore, the present invention aims at providing a molecule or acomposition as described above for preventing or treating septic shock,cachexia, immune diseases such as multiple sclerosis and Crohn's diseaseand skin disorders such as bullous, inflammatory and neoplasticdermatosis.

Finally, the present invention aims at providing a molecule as describedabove for determining interferon gamma levels in a sample.

All the aims of the present invention are considered to have been met bythe embodiments as set out below.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 schematically shows 2 different tetravalent antibody constructs(MoTAB I and MotabII). MoTAb I represents a molecule which consists of 4identical scFv's in the format of a homodimer of 2 identical molecules,each containing 2 scFv's. MoTAb II represents a full-size antibodymolecule to which 2 scFv's with the same specificity are attached at thecarboxyterminus. Optionally, these constructs contain apurification/detection tag.

See also further Example 4.

FIG. 2 shows the coding (SEQ ID NO 1) and amino acid sequence (SEQ ID NO2) of humanized D9D10 scFv (containing a C-terminal 6-histidine tag(bold)). CDR regions are underlined. Mutations (murine—>human) are boldand underlined. The N-terminal pelB signal sequence is put in bold.

FIGS. 3 and 4 shows the binding of different concentrations of murinescFvD9D10 (FIG. 3) and humanized scFvD9D10 (FIG. 4) to human IFNγ. HumanIFNγ is immobilized indirectly to the CM5 sensorchip via the murineD9D10 full size antibody as described in example 1. Association rateconstants derived from these binding curves are shown. Dissociation rateconstants could not be measured accurately as dissociation is hardlydetectable (<5×10⁴ s⁻¹) in this experimental setup.

FIG. 5 shows a schematic representation of the mammalian expressionplasmid pEE12hD9D10 used for expression of humanized D9D10 wholeantibody in (1) COS cells (2) stable recombinant Ns0 cell lines.

Major Plasmid Building Blocks:

prokaryotic sequences for plasmid DNA preparation in E.coli (ori ofreplication and amp^(R) ampicilline resistance expression unit)

SV40 origin of replication (part of SV40E, SV40 early promoter) allowingtransient expression in SV40 permissive, T-antigen producing cell lines(e.g. COS)

human Cytomegalovirus major immediate early promoter/enhancer(hCMVprom+intron) controlled expression casette for hD9D10 heavy chainprotein (hD9D10-H)

human Cytomegalovirus major immediate early promoter/enhancer(hCMVprom+intron) controlled expression casette for hD9D10 light chainprotein (hD9D10-L)

SV40 early promoter (SV40E) controlled glutamine synthetase cDNA (GS)expression unit for selection/amplification

polyA=SV40 early region poly-adenylation signal

intron +polyA=SV40 t-antigen intron+SV40 early region poly-adenylationsignal

FIG. 6 shows a schematic representation of the mammalian expressionplasmid pEE14hD9D10 used for expression of humanized D9D10 wholeantibody in (1) COS cells (2) stable recombinant CHO-K1 cell lines.

Major Plasmid Building Blocks:

prokaryotic sequences for plasmid DNA preparation in E.coli (on ofreplication and amp^(R) ampicilline resistance expression unit)

SV40 origin of replication (part of SV40E, SV40 early promoter) allowingtransient expression in SV40 permissive, T-antigen producing cell lines(e.g. COS)

human Cytomegalovirus major immediate early promoter/enhancer(hCMVprom+intron) controlled expression casette for hD9D10 heavy chainprotein (hD9D10-H)

human Cytomegalovirus major immediate early promoter/enhancer(hCMVprom+intron) controlled expression casette for hD9D10 light chainprotein (hD9D10-L)

SV40 late promoter (SV40L) controlled glutamine synthetase mini gene(GS+intron) expression unit for selection/amplification

polyA=SV40 early region poly-adenylation signal

intron+polyA=SV40 t-antigen intron+SV40 early region poly-adenylationsignal

FIG. 7 shows the cDNA sequence encoding the humanized D9D 10 heavy chainfusion protein.

bp 1-60 : D9D10 Kappa-light chain signal sequence

bp 61-411: humanized D9D10 heavy chain variable domain

bp 412-1401: human IgG1 heavy chain constant domain(C_(H)1-Hinge-C_(H)2-C_(H)3)

bp 1402-1404: leu codon added by PCR cloning strategy (SEQ ID NO 66)

FIG. 8 shows the cDNA sequence encoding the humanized D9D10 and MoTAbIIlight chain fusion protein.

bp 1-60 : D9D10 Kappa-light chain signal sequence

bp 61-381: humanized D9D10 light chain variable domain

bp 382-699: human kappa light chain constant domain (SEQ ID NO 68)

FIG. 9 shows the amino acid sequence of the humanized D9D10 heavy chainfusion protein.

Aa 1-20: D9D10 light chain signal sequence

Aa 21-137: humanized heavy chain variable domain of D9D10

Aa138-467: human IgG1 heavy chain constant domain(C_(H)1-hinge-C_(H)2-C_(H)3)

Aa 468: leu added by PCR cloning strategy

Aa 351: pro was mutated to ser: inactivation C1q complement binding

Number of residues: 468.

Molecular weight (MW): 51413 (SEQ ID NO 67)

FIG. 10 shows the amino acid sequence of the humanized D9D10 and MoTAbIIlight chain fusion protein.

Aa 1-20: D9D10 light chain signal sequence

Aa 21 -127: humanized light chain variable domain of D9D10

Aa 128-233: human kappa light chain constant domain

Number of residues: 233.

Molecular weight (MW): 25582 (SEQ ID NO 69)

FIG. 11 shows the binding in ELISA of different concentrations ofhumanized D9D10 and humanized D9D10 MoTAbII (=different dilutions ofcrude COS supernatant containing humanized D9D10 or humanized D9D10MoTAbII) to immobilized human IFNγ. The assay is performed as describedin example 2.

FIG. 12 shows the interaction of humanized D9D10 (=crude COS supernatantcontaining humanized D9D10) with IFNγ using SPR analysis. The assay isperformed as described in example 2.

FIG. 13 shows the binding in ELISA of different concentrations ofpurified humanized D9D10 and MoTAbII to immobilized human IFNγ. Theassay is performed as described in example 2.

FIG. 14 shows a schematic representation of the expressionplasmidpMoTAbIH6 used for the expression of MoTAbI in E. coli.

FIG. 15 shows the cDNA sequence of MoTAbI

bp 1-351:V_(H) D9D10

bp 352 -396: (G₄S)₃ linker

bp 397 -717: V_(L) D9D10

bp 718 -750: human IgG3 upper hinge

bp 751 -855: helix-turn-helix dimerisation domain

bp 856 -888: human IgG3 upper hinge

bp 889 -1239 V_(H) D9D10

bp 1240 -1284: (G₄S)₃ linker

bp 1285 -1605: V_(L) D9D10

bp 1606 -1623: His6 tag (SEQ ID NO 84)

FIG. 16 shows the AA sequence of MoTAbI

aa 1-117: V_(H) D9D10

aa 118 -132: (G₄S)₃ linker

aa 133 -239: V_(L) D9D10

aa 240 -250: human IgG3 upper hinge

aa 251 -285: helix-turn-helix dimerisation domain

aa 286 -296: human IgG3 upper hinge

aa 297 -413: V_(H) D9D10

aa 414 -428: (G₄S)₃ linker

aa 429 -525: V_(L) D9D10

aa 526 -531: His6 tag (SEQ ID NO 85)

FIG. 17 shows a schematic representation of the mammalian expressionplasmid pEE12MoTAbII used for expression of D9D10MoTAbII recombinantantibody in (1) COS cells (2) stable recombinant Ns0 cell lines.

Major Plasmid Building Blocks:

prokaryotic sequences for plasmid DNA preparation in E.coli (ori ofreplication and amp^(R) ampicilline resistance expression unit)

SV40 origin of replication (part of SV40E, SV40 early promoter) allowingtransient expression in SV40 permissive, T-antigen producing cell lines(e.g. COS)

human Cytomegalovirus major immediate early promoter/enhancer(hCMVprom+intron) controlled expression casette for D9D10MoTAbII heavychain protein (MoTAbII-H)

human Cytomegalovirus major immediate early promoter/enhancer(hCMVprom+intron) controlled expression casette for D9D10MoTAbII lightchain protein (MoTAbII-L)

SV40 early promoter (SV40E) controlled glutamine synthetase cDNA (a)expression unit for selection/amplification

polyA=SV40 early region poly-adenylation signal

intron+polyA=SV40 t-antigen intron+SV40 early region poly-adenylationsignal

FIG. 18 shows a schematic representation of the mammalian expressionplasmid pEE14MoTAbII used for expression of D9D10MoTAbII recombinantantibody in (1) COS cells (2) stable recombinant CHO-K1 cell lines.

Major Plasmid Building Blocks:

prokaryotic sequences for plasmid DNA preparation in E.coli (ori ofreplication and amp^(R) ampicilline resistance expression unit)

SV40 origin of replication (part of SV40E, SV40 early promoter) allowingtransient expression in SV40 permissive, T-antigen producing cell lines(e.g. COS)

human Cytomegalovirus major immediate early promoter/enhancer(hCMVprom+intron) controlled expression casette for D9D10MoTAbII heavychain protein (MoTAbII-H)

human Cytomegalovirus major immediate early promoter/enhancer(hCMVprom+intron) controlled expression casette for D9D10MoTAbII lightchain protein (MoTAbII-L)

SV40 late promoter (SV40L) controlled glutamine synthetase mini gene(GS+intron) expression unit for selection/amplification

polyA=SV40 early region poly-adenylation signal

intron+polyA=SV40 t-antigen intron+SV40 early region poly-adenylationsignal

FIG. 19 shows the cDNA sequence encoding the MoTABII fusion protein

bp 1-60: D9D10 Kappa-light chain signal sequence

bp 61-411: humanized D9D10 heavy chain variable domain

bp 412-1401: human IgG1 heavy chain constant domain(C_(H)1-Hinge-C_(H)2-C_(H)3)

bp 1402-1404: leu codon added by PCR cloning strategy

bp 1405-1416: gly(3)-ser codon

bp 1417-2133: humanized D9D10 ScFv (SEQ ID NO 89)

FIG. 20 shows the amino acid sequence of MoTABII fusion protein

Aa 1-20: mouse D9D10 light chain signal sequence

Aa 21-137: humanized heavy chain variable domain of D9D10

Aa 138-467: human IgG1 heavy chain constantdomain(C_(H)1-hinge-C_(H)2-C_(H)3)

Aa 351: pro mutated to ser: inactivation C1q complement binding

Aa 468: leu added by cloning strategy

Aa 469-472: gly(3)-ser linker

Aa 473-711: humanized D9D10 ScFv (V_(H)473-490/gly-serlinker/V_(L)605-711) (SEQ ID NO 90)

FIG. 21 shows the interaction of MoTAbII (=crude COS supernatantcontaining MoTAbII) with IFNγ using SPR analysis. The assay is performedas described in example 4.

FIG. 22 shows the amino acid sequence of the D9D10L10 diabody

aa 1-117: V_(H) D9D10

aa 118 -127: (G₄S)₂ linker

aa128-234: V_(L) D9D10

aa 235 -240: His6-tag (SEQ ID NO 91)

FIG. 23 shows the coding sequence of the D9D10L10 diabody

bp 1-351: V_(H) D9D10

bp 352 -381: (G₄S)₂ linker

bp 382 -702: V_(L) D9D10 (SEQ ID NO 92)

FIG. 24 shows the amino acid sequence of the D9D10 L5 diabody

aa 1-117: V_(H) D9D10

aa 118 -122: G₄S linker

aa 123 -229: V_(L) D9D10

aa 230 -235: His6-tag 5 (SEQ ID NO 93)

FIG. 25 shows the coding sequence of the D9D10 L5 diabody

bp 1-351: V_(H) D9D10

bp 352 -366: G₄S linker

bp 367 -687: V_(L) D9D10 (SEQ ID NO 94)

FIG. 26 shows the interaction of humanized L5 D9D10 diabody (=crudelysate from E. Coli) with IFNγ using SPR analysis. The assay isperformed as described in example 5.

FIG. 27 shows the coding sequence of the D9D10 L0 triabody

bp 1-351: V_(H) D9D10

bp352-672: V_(L) D9D10 (SEQ ID NO 101)

FIG. 28 shows the amino acid sequence of the D9D10 L0 triabody

aa 1-117: V_(H) D9D10

aa 118 -224: V_(L) D9D10

aa 225 -230: His6-tag (SEQ ID NO 102)

FIG. 29 shows the interaction of humanized L0 D9D10 triabody (=crudelysate from E. Coli) with IFNγ using SPR analysis. The assay isperformed as described in example 6.

FIG. 30 shows the neutralization of IFN-gamma-induced MHC class IIupregulation on human primary keratinocytes by D9D10 or D9D10 scFv.Human keratinocytes were cultured for 24 h with or without (not shown)100 U/ml huIFN-gamma in the absence or the presence of D9D10 (2 μg/ml).Resting human keratinocytes do not express MHC class II. IFN-gammainduces expression of MHC class II in the keratinocytes and D9D10 (upperpanel) or scFv D9D10 (lower panel) inhibit this IFN-gamma-induced MHCclass II expression. See also further Example 7.1.

FIG. 31 shows the neutralization of IFN-gamma-induced MHC class IIupregulation on human primary keratinocytes by crude COS supernatantcontaining either humanized D9D10 or MoTAbII. The experiment wasperformed as described in FIG. 30 thin line: human keratinocytes treatedwith human IFNγ

bold line: A: human keratinocytes not treated with human IFNγ

B: effect of 400 ng/ml murine D9D10

C: effect of humanized D9D10 (crude COS supernatant)

D: effect of MoTAbII (crude COS supernatant)

FIG. 32 shows the effect of the anti-IFN-gamma antibody F3 and scFvF3 onthe survival of mice in which the lethal shock syndrome called“Shwartzman reaction” is induced. See also further Example 7.3.

FIG. 33 shows the effect of the anti-IFN-gamma antibody F3 and scFvF3 onbody weight of mice exhibiting IFN-gamma induced cachexia. Mortality(number of dead mice/total number of mice) is shown between brackets andthe symbol “+”. See also further Example 7.4.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The invention described herein draws on previously published work andpending patent applications. By way of example, such work consists ofscientific papers, patents or pending patent applications. All of thesepublications and applications, cited previously or below are herebyincorporated by reference.

The present invention is based on the finding that a molecule whichbinds and neutralizes human interferon-gamma and which is chosen fromthe group consisting of:

-   -   a scFv comprising the humanized variable domain of the        monoclonal antibody D9D10    -   a chimeric antibody comprising the humanized variable domain of        the monoclonal antibody D9D10    -   a diabody comprising the humanized variable domain of the        monoclonal antibody D9D10    -   a multivalent antibody    -   a ruminant antibody    -   is useful to treat diseases where IFNγ activity is pathogenic.

As used herein the terms “molecule which binds and neutralizes IFNγ”refer to a molecule which recognizes and binds any particular epitope ofIFNγ resulting in the neutralization of any bioactivity of IFNγ.Particular epitopes of IFNγ relate to the so-called E2 epitoperecognized and bound by the mAb D9D10, the so-called E1 epitope (Kwok etal., 1993) or any other epitope. IFNγ specifically relates to human IFNγbut may also relate to non-human primate, mouse, rat, sheep, goat,camel, cow, llama or any other IFNγ. Furthermore, the term “bioactivityof IFNγ” relates to the antiviral activity (Billiau, 1996), theinduction of the expression of MHC-class-II molecules by macrophages andother cell types (Steinman et al., 1980), the stimulation of theproduction of inflammatory mediators such as TNFα, IL-1 and NO (Lorsbachet al., 1993), the induction of the expression of adhesion moleculessuch as ICAM-1 (Dustin et al., 1988) and of important costimulators suchas the B7 molecules on professional antigen presenting cells (Freedmanet al., 1991), the induction of macrophages to become tumoricidal (Paceet al., 1983), the induction of Ig isotype switching (Snapper and Paul,1987), any pathological and/or clinical activity during diseases whereIFNγ is pathogenic (Billiau, 1996) or any other known bioactivity ofIFNγ. In this regard, it should be clear that any assay systemdemonstrating the IFNγ-neutralizing capacity of a molecule, such as theones described by Novelli et al. (1991), Lewis (1995) and Turano et al.(1992) can be used. Some of these assays are also described in thesubsection Evaluation of anti-IFNγ neutralizing molecules in theExamples section of the present application (see further). It should benoted that the molecules which bind and neutralize IFN-γ as describedabove neutralize at least one bioactivity, but not necessarily allbioactivities, of IFN-γ.

The present invention further relates to a scFv comprising the humanizedvariable domain of the monoclonal antibody D9D10. As used herein, theterm single-chain Fv, also termed single-chain antibody, refers toengineered antibody constructs prepared by isolating the binding domains(both heavy and light chain) of a binding antibody, and supplying alinking moiety which permits preservation of the binding function. Thisforms, in essence, a radically abbreviated antibody, having only thevariable domain necessary for binding the antigen. Determination andconstruction of single chain antibodies are described in U.S. Pat. No.4,946,778 to Ladner et al. and in the Examples section of the presentapplication (see further). The term “humanized” means that at least aportion of the framework regions of an immunoglobulin or engineeredantibody construct is derived from human immunoglobulin sequences. Itshould be clear that any method to humanize antibodies or antibodyconstructs, as for example by variable domain resurfacing as describedby Roguska et al. (1994) or CDR grafting or reshaping as reviewed byHurle and Gross (1994), can be used. The humanization of the scFvcomprising the variable domain of the monoclonal antibody D9D10 isdescribed further in the Examples section of the present application.The monoclonal antibody D9D10 was prepared essentially as described bySandvig et al. (1987) and Froyen et al. (1993). It should also be notedthat the process of humanization of an antibody or antibody construct isregularly accompanied by a significant loss in binding affinity of thisantibody or antibody construct (Kettleborough et al., 1991; Park et al.,1996 and Mateo et al., 1997). In contrast, and surprisingly, theconstructs humanized by the present inventors were not characterized bya significant loss in binding affinity in comparison to theirnon-humanized counterparts.

The present invention also relates to a chimeric antibody comprising thehumanized variable domain of the monoclonal antibody D9D10. The term“chimeric antibody” refers to an engineered antibody constructcomprising variable domains of one species (such as mouse, rat, goat,sheep, cow, llama or camel variable domains), which may be humanized ornot, and constant domains of another species (such as non-human primateor human constant domains) (for review see Hurle and Gross (1994)). Itshould be clear that any method known in the art to develop chimericantibodies or antibody constructs can be used. The generation of achimeric antibody comprising the humanized variable domain of themonoclonal antibody D9D10 is described further in the Examples sectionof the present application.

The present invention also concerns a diabody comprising the humanizedvariable domain of the monoclonal antibody D9D10. The term “diabody”relates to two non-covalently-linked scFv's, which then form a so-calleddiabody, as described in detail by Holliger et al. (1993) and reviewedby Poljak (1994). It should be clear that any method to generatediabodies, as for example described by Holliger et al. (1993), Poljak(1994) and Zhu et al. (1996), can be used. The generation of diabodiescomprising the variable domain of the monoclonal antibody D9D10 isdescribed further in the Examples section of the present application.

It should also be clear that the scFv's, chimeric antibodies anddiabodies described above are not limited to comprise the variabledomain of the monoclonal antibody D9D10 but may also comprise variabledomains of other anti-IFNγ antibodies, such as the sheep anti-IFNγantibody described further in the present application, which efficientlyneutralize the bioactivity of IFNγ.

Furthermore, the diabodies described above may also comprise two scFv'sof different specificities. For example, the latter diabodies maysimultaneously neutralize IFNγ on the one hand and may target anothermolecule, such as TNF-α, IL-1, IL-2, B7.1 or CD80, B7.2 or CD86, IL-12,IL-4, IL-10, CD40, CD40L, IL-6, tumour growth factor-beta (TGF-β),transferrin receptor, insulin receptor and prostaglandin E2 or any othermolecule, on the other hand.

The present invention also concerns multivalent antibodies which bindand neutralize IFNγ. As used herein, the term multivalent antibodyrefers to any IFNγ-binding and IFNγ-neutralizing molecule which has morethan two IFNγ-binding regions. Examples of such multivalent antibodiesare triabodies, tetravalent antibodies, peptabodies and hexabodies whichbind and neutralize IFNγ and which have three, four, five and sixIFNγ-binding regions, respectively.

The present invention thus relates, as indicated above, to triabodieswhich bind and neutralize IFNγ. As used herein, the term “triabody”relates to trivalent constructs comprising 3 scFv's, and thus comprising3 variable domains, as described by Kortt et al. (1997) and Iliades etal. (1997). A method to generate triabodies is described by Kortt et al.(1997) and the generation of triabodies comprising the variable domainof the monoclonal antibody D9D10 is described further in the Examplessection of the present application. It should be noted that thetriabodies of the present invention may comprise: 3 variable domains of3 different anti-IFNγ Ab's (i.e. 3 anti-IFNγ Ab's which recognize andbind a different epitope on IFNγ [see also above]), 3 variable domainsof 3 identical anti-IFNγ Ab's such as 3 variable domains of D9D10 or 3variable domains of humanized D9D10 or 3 variable domains of sheepanti-IFNγ Ab's or 3 humanized variable domains of sheep anti-IFNγ Ab's,1 or 2 variable domain(s) of anti-IFNγ Ab's in combination with 2 or 1variable domain(s) of an Ab which binds to any other molecule than IFNγ,respectively. Examples of such other molecules comprise TNF-α, IL-1,IL-2, B7.1 or CD80, B7.2 or CD86, IL-12, IL-4, IL-10, CD40, CD40L, IL-6,tumour growth factor-beta (TGF-β), transferrin receptor, insulinreceptor and prostaglandin E2.

The present invention further relates to tetravalent antibodies whichbind and neutralize IFNγ. As used herein, the term “tetravalentantibody” refers to engineered antibody constructs comprising 4antigen-binding regions as described by Pack et al. (1995) and Coloma &Morrison (1997). Methods to generate these tetravalent antibodyconstructs are also described by the latter authors. The generation ofthe following 2 different tetravalent antibodies comprising the variabledomain of the monoclonal antibody D9D10 are described further in theExamples section of the present application: MoTabI which consists of 4identical humanized D9D10 scFv's in the format of a homodimer of twoidentical molecules each containing two D9D10 scFv's which are linkedtogether using a dimerization domain; the latter domain also drives thehomodimerization of the molecule, and, MoTab II which consists of afull-size humanized D9D10 molecule to which two humanized D9D10 scFv'sare attached at the carboxyterminus (CH3-domain). It should be notedthat the tetravalent antibodies of the present invention may comprise: 4variable domains of 4 different anti-IFNγ Ab's (i.e. anti-IFNγ Ab'swhich recognize and bind to a different epitope on IFNγ), 4 variabledomains of 4 identical anti-IFNγ Ab's such as 4 variable domains ofD9D10 or 4 variable domains of humanized D9D10 or 4 variable domains ofsheep anti-IFNγ Ab's or 4 humanized variable domains of sheep anti-IFNγAb's, 2 variable domain(s) of one anti-IFNγ Ab in combination with 2variable domain(s) of another anti-IFNγ Ab, 2 variable domain(s) ofanti-IFNγ Ab's in combination with 2 variable domain(s) which binds toany other molecule than IFNγ. Examples of such other molecules compriseTNF-α, IL-1, IL-2, B7.1 or CD80, B7.2 or CD86, IL-12, IL-4, IL-10, CD40,CD40L, IL-6, TGF-β, transferrin receptor, insulin receptor andprostaglandin E2.

Furthermore, the term “dimerization domain” of MoTab I refers to anymolecule known in the art which is capable of coupling the two identicalmolecules. Examples of such domains are the leucine zipper domain (deKruif & Logtenberg, 1996), the helix-turn-helix motif described by Packet al. (1993), the max-interacting proteins and related molecules asdescribed in U.S. Pat. No. 5,512,473 to Brent & Zervos and thepolyglutamic acid-polylysine domains as described in U.S. Pat. No.5,582,996 to Curtis.

The present invention thus relates, as indicated above, to peptabodiesand hexabodies which bind and neutralize IFNγ. As used herein, the term“peptabodies” relates to pentavalent constructs as described in detailby Terskikh et al. (1997). The term “hexabodies” relates to hexavalentconstructs which are similar to the pentavalent constructs as describedin detail by Terskikh et al. (1997) but wherein the pentamerizationdomain is replaced by any hexamerization domain known in the art. Amethod to generate peptabodies is also described by Terskikh et al.(1997) and a method to generate hexabodies can be derived from thedescription by the latter authors. It should be noted that thepeptabodies and hexabodies of the present invention may comprise: 5(relating to the peptabodies) or 6 (relating to the hexabodies) variabledomains of 5 or 6 different anti-IFNγ Ab's (i.e. 5 or 6 anti-IFNγ Ab'swhich recognize and bind a different epitope on IFNγ [see also above]),5 or 6 variable domains of identical anti-IFNγ Ab's such as 5 or 6variable domains of D9D10, or, 5 or 6 variable domains of humanizedD9D10, or, 5 or 6 variable domains of sheep anti-IFNγ Ab's, or, 5 or 6humanized variable domains of sheep anti-IFNγ Ab's, less than 5 or 6variable domain(s) of any anti-IFNγ Ab's in combination with less than 5or 6 variable domain(s) of an Ab which binds to any other molecule thanIFNγ, respectively. Examples of such other molecules comprise TNF-α,IL-1, IL-2, B7.1 or CD80, B7.2 or CD86, IL-12, IL-4, IL-10, CD40, CD40L,IL-6, TGF-β, transferrin receptor, insulin receptor and prostaglandinE2.

The present in invention further relates to ruminant antibodies whichbind and neutralize IFNγ. The term “ruminant” relates to animalsbelonging to the suborder Ruminantia of even-toed hoofed mammals (assheep, goats, cows, giraffes, deer, llama, vicunas and camels) that chewthe cud and have a complex 3- or 4-chambered stomach.

More specifically, the present invention relates to sheep antibodieswhich bind and neutralize IFNγ. The term “sheep” relates to any ofnumerous ruminant mammals belonging to the genus Ovis. The generation ofsheep anti-IFNγ antibodies is described in the Examples section of thepresent application. The present invention also relates to sheepmonoclonal antibodies. As used herein, the term “monoclonal antibody”refers to an antibody composition having a homogeneous antibodypopulation. The term is not limited regarding the species or source ofthe antibody, nor is it intended to be limited by the manner in which itis made. Indeed, the monoclonal sheep antibodies of the presentinvention can be generated by any method known in the art. It should benoted that also humanized antibodies, scFv's or any other fragmentthereof which has largely retained the specificity of said sheepantibody or sheep monoclonal antibody are covered by the presentinvention. As used herein, the term “fragment” refers to F(ab), F(ab′)2,Fv, and other fragments which retain the antigen binding function andspecificity of the parent antibody. It should also be understood thatthe variable domains of the sheep anti-IFNγ (monoclonal) antibodies orscFv of the sheep anti-IFNγ (monoclonal) antibodies may be part of thechimeric antibodies, diabodies, triabodies, tetravalent antibodies,peptabodies and hexabodies as described above.

The present invention further relates to scFv's, chimeric antibodies,diabodies, triabodies, tetravalent antibodies, peptabodies, hexabodiesand sheep antibodies which bind and neutralize IFNγ and which areproduced by the methods as described above and in the Examples sectionof the present application.

The present invention further relates to a composition comprising scFv'sand/or chimeric antibodies and/or diabodies and/or triabodies and/ortetravalent antibodies and/or peptabodies and/or hexabodies and/or sheepantibodies which bind and neutralize IFNγ in a pharmaceuticallyacceptable excipient, possibly in combination with other drugs or otherantibodies, antibody derivatives or constructs for use as a medicamentto prevent or treat septic shock, cachexia, immune diseases such asmultiple sclerosis and Crohn's disease and skin disorders such asbullous, inflammatory and neoplastic dermatoses. Examples of such otherdrugs or other antibodies, antibody derivatives or constructs are, withregard to septic shock: an isotonic crystalloid solution such as saline,dopamine, adrenaline and antibiotics; with regard to cachexia:anti-TNF-alpha antibodies; with regard to multiple sclerosis: ACTH andcorticosteroids, interferon beta-1b (Betaseron), interferon beta-1a(Avonex), immunosuppressive drugs such as azathioprine, methotrexate,cyclophosphamide, cyclosporin A and cladribine (2-CdA), copolymer 1(composed of 4 amino acids common to myelin basic proteins), myelinantigens, roquinimex A, the mAb CAMPATH-1H and potassium channelblockers; with regard to Crohn's disease: sulfasalazine,corticosteroids, 6 mercaptopurine/azathioprine and cyclosporin A; withregard to psoriasis: cyclosporin A, methotrexate, calcipotriene(Dovonex), zidovudine (Retrovir), histamine2 receptor antagonists suchas ranitidine (Zantac) and cimetidine (Tagamet), propylthiouracil,acitretin (Soriatane), fumaric acid, vitamin D derivates, tazarotene(Tazorac), IL-2 fusion toxin, tacrolimus (Prograf), CTLA4Ig, anti-CD4mAb's and T-cell receptor peptide vaccines. It should also be clear thatany possible mixture of the above-indicated IFN-γ-binding molecules maybe part of the above-indicated pharmaceutical composition.

As used herein, the term “composition” refers to any compositioncomprising as an active ingredient scFv's and/or chimeric antibodiesand/or diabodies and/or triabodies and/or tetravalent antibodies and/orpeptabodies and/or hexabodies and/or sheep antibodies which bind andneutralize IFNγ according to the present invention possibly in thepresence of suitable excipients known to the skilled man. The scFv'sand/or chimeric antibodies and/or diabodies and/or triabodies and/ortetravalent antibodies and/or peptabodies and/or hexabodies and/or sheepantibodies which bind and neutralize IFNγ of the invention may thus beadministered in the form of any suitable composition as detailed belowby any suitable method of administration within the knowledge of askilled man. The preferred route of administration is parenterally. Inparenteral administration, the compositions of this invention will beformulated in a unit dosage injectable form such as a solution,suspension or emulsion, in association with a pharmaceuticallyacceptable excipient. Such excipients are inherently nontoxic andnontherapeutic. Examples of such excipients are saline, Ringer'ssolution, dextrose solution and Hank's solution. Nonaqueous excipientssuch as fixed oils and ethyl oleate may also be used. A preferredexcipient is 5% dextrose in saline. The excipient may contain minoramounts of additives such as substances that enhance isotonicity andchemical stability, including buffers and preservatives.

The scFv's and/or chimeric antibodies and/or diabodies and/or triabodiesand/or tetravalent antibodies and/or peptabodies and/or hexabodiesand/or sheep antibodies which bind and neutralize IFNγ of the inventionare administered at a concentration that is therapeutically effective totreat or prevent septic shock, cachexia, immune diseases such asmultiple sclerosis and Crohn's disease and skin disorders such asbullous, inflammatory and neoplastic dermatoses. The dosage and mode ofadministration will depend on the individual. Generally, thecompositions are administered so that the scFv's and/or chimericantibodies and/or diabodies and/or triabodies and/or tetravalentantibodies and/or peptabodies and/or hexabodies and/or sheep antibodieswhich bind and neutralize IFNγ are given at a dose between 1 μg/kg and10 mg/kg, more preferably between 10 μg/kg and 5 mg/kg, most preferablybetween 0.1 and 2 mg/kg for each IFN-γ-binding molecule. Preferably,they are given as a bolus dose. Continuous short time infusion (during30 minutes) may also be used. If so, the scFv's and/or chimericantibodies and/or diabodies and/or triabodies and/or tetravalentantibodies and/or peptabodies and/or hexabodies and/or sheep antibodieswhich bind and neutralize IFNγ or compositions comprising the same maybe infused at a dose between 5 and 20 μg/kg/minute, more preferablybetween 7 and 15 μg/kg/minute (for each IFN-γ-binding molecule).

According to the specific case, the “therapeutically effective amount”of a scFv's and/or chimeric antibodies and/or diabodies and/ortriabodies and/or tetravalent antibodies and/or peptabodies and/orhexabodies and/or sheep antibodies which bind and neutralize IFNγ neededshould be determined as being the amount sufficient to cure the patientin need of treatment or at least to partially arrest the disease and itscomplications. Amounts effective for such use will depend on theseverity of the disease and the general state of the patient's health.Single or multiple administrations may be required depending on thedosage and frequency as required and tolerated by the patient.

The present invention further relates to scFv's and/or chimericantibodies and/or diabodies and/or triabodies and/or tetravalentantibodies and/or peptabodies and/or hexabodies and/or sheep antibodieswhich bind and neutralize IFNγ for determining IFNγ levels in abiological sample, comprising:

-   -   1) contacting the biological sample to be analysed for the        presence of IFNγ with a scFv and/or chimeric antibody and/or        diabody and/or triabody and/or tetravalent antibody and/or        peptabodies and/or hexabodies and/or sheep antibody as defined        above,    -   2) detecting the immunological complex formed between IFNγ and        said scFv and/or chimeric antibody and/or diabody and/or        triabody and/or tetravalent antibody and/or peptabodies and/or        hexabodies and/or sheep antibody.

As used herein, the term “a method to detect” refers to any immunoassayknown in the art such as assays which utilize biotin and avidin orstreptavidin, ELISA's and immunoprecipitation, immunohistochemicaltechniques and agglutination assays. A detailed description of theseassays is given in WO 96/13590 to Maertens & Stuyver. Theimmunohistochemical detection of IFNγ in cryosections of spinal cord andbrain of non-human primates suffering from experimental autoimmuneencephalomyelitis is described in detail in the Examples section of thepresent application. The term “biological sample” relates to anypossible sample taken from a mammal including humans, such as blood(which also encompasses serum and plasma samples), sputum, cerebrospinalfluid, urine, lymph or any possible histological section, wherein IFNγmight be present.

The present invention will now be illustrated by reference to thefollowing examples which set forth particularly advantageousembodiments. However, it should be noted that these embodiments areillustrative and are not to be construed as restricting the invention inany way.

EXAMPLES

1. Generation of Humanized scFvD9D10

As the use of mouse monoclonals in humans induces a HAMA response, ahumanized antibody or antibody derivative is the alternative. HumanizedscFvD9D10 need to have similar binding and neutralization properties astheir original mouse counterparts, but will elicit hardly any immuneresponse in humans as compared to the parent mouse scFv.

1.1. Modelling

We used computer modelling techniques for the construction of ahumanized scFvD9D10 in order to develop an active scFv with retainedstructure and affinity. The scFv was humanized using a resurfacingstrategy which includes the replacement of ‘non-human’ residues withoutsignificant structural changes of the scFv molecule. This work consistedof 2 main parts. In the first part, a 3D-structure of the mouse scFv wasconstructed. For this purpose, we have homology-modeled D9D10 using IgV_(L) and V_(H) domains with a similar sequence and a known structure.In the second part (the actual humanization step), we have aligned D9D10with similar human sequences to identify ‘typically human residues’.After verifying their structural compatibility with the D9D10model, theyhave been proposed as residues-to-be-humanized.

PART 1: 3D-structure of scFvD9D10

Identification of Known Structures with the Most Resembling Sequence

Different BLAST-searches were performed by entering the D9D10 sequenceof either V_(K) or V_(H), by using the ‘BLASTP’ search program and byselecting the Brookhaven Protein Data Bank as the database to besearched. This search was performed 4 times, namely for V_(K) with andwithout CDR-loops and for V_(H) with and without CDR-loops. The obtaineddata are summarized in Table 1. TABLE 1 Summary of BLAST-search resultsPDB score + CDR score − CDR rank rank Code ident./sim. ident./sim. forV_(H) source I.D. A) BLAST-search using D9D10-V_(K) sequence 1 1BAF87%/92% 90%/95% >50 mouse Fab frag. mAb An02 compl. w. its hapten(2,2,6,6-Tetramethyl-1- Piperidinyloxy-Dinitrophenyl) 2 1FOR 80%/90%85%/93% 16 mouse Igg2a Fab frag. (Fab17-Ia) 3 2IFF 78%/86% 84%/90% 15mouse Igg1 Fab Frag. (Hyhel-5) compl. w. Chicken Lysozyme mutant R68K 41FIG 75%/86% 80%/90% 28 mouse Chain L, Immunogl G1 (Kappa Light Chain)Fab' frag, Mouse 5 1FVB 80%/87% 83%/89% >50 mouse IgA Fv frag.(Anti-Alpha(1->6) Dextran) (Theoret. Model) 6 2HFL 77%/85% 83%/89% 14mouse IgGl Fab frag. (HyHEL-5) compl. w. Chicken Lysozyme . . . . . . .. . . . . . . . . . . . . . 19  1NCA 60%/73% 70%/84% 1 mouse N9neuraminidase-NC41 compl. w. Influenza Virus . . . . . . . . . . . . . .. . . . . . . B) BLAST-search using D9D10-V_(H) sequence 1 1NCA 83%/89%91%/95% 19 mouse? N9 neuraminidase-NC41 compl. w. Influenza Virus 2 1NCB80%/88% 87%/94% >50 mouse? N9 Neuraminidase-Nc41 Mut. N329D compl. w.Fab, Influenza Virus 3 1TET 80%/86% 87%/92% 38 mouse Igg1 Monocl. Fabfrag (Te33) compl. w. Cholera Toxin Peptide 3 4 1DBA 80%/87% 86%/92% >50mouse Fab' frag. of the Db3 Anti- Steroid Monocl. Ab . . . . . . . . . .. . . . . . . . . . . 16  1FOR 58%/76% 63%/83% 2 mouse Igg2a Fab frag.(Fab17-Ia) . . . . . . . . . . . . . . . . . . . . .

A sequence similarity of more than 70% guarantees a strong structuralsimilarity. For V_(K), at least 6 very good matching structures (allmurine proteins) could be identified: 1BAF, 1FOR, 2IFF, 1FIG, 1FVB and2HFL. The scores for the search with CDR-loops varied from 87% to 77%for identical residues, and from 92% to 85% for chemically similarresidues. The scores for the search without CDR-loops ranged from 90% to83% identical residues and from 95% to 89% similar residues. The smalldifference in homology between the searches with and without CDR-loopssuggests that even some of the CDR-loops are structurally similar. ForV_(H), analogous results were obtained. Four very well matchingstructures could be identified: 1NCA, 1NCB, 1TET and 1DBA with scoresvarying from 83% to 80% identical residues and from 89% to 87% similarresidues when CDR-loops are included. If CDR-loops were not taken intoaccount, significantly higher scores were obtained: from 91% to 86% foridentical residues and 95% to 92% for similar residues. The latter wasdue to the fact that CDR-H3 from D9D10 was not matching well with anysequence.

Three-Dimensional Fitting of the Best Candidates

From these scores, it was clear that the V_(K)-fragment from 1FORresembled very well V_(K) from D9D10 (rank nr 2). A reasonably wellhomology was also found for its V_(H) counterpart (rank nr 16). For theheavy domain, 1NCA had a very high score for V_(H) (rank nr 1) and anacceptable score for its V_(K)-domain (rank nr 19). Since the β-barrelsof Fv fragments are well conserved, and since for both V_(K) and V_(H)we dispose of two very good resembling fragments with fairly wellmatching counterparts, we had enough information to start theconstruction of the D9D10 model.

When superimposing (fitting) the complete main chain of 1FOR and 1NCA weobtained a root-mean-square (rms) deviation of 1.1 Å (values around. orless than 1 Å indicate a strong structural similarity). Fitting on V_(K)alone gave 1.0 Å and on V_(H) we obtained 0.8 Å. This means that boththe complete structures and the separate V-domains are nearly identical.In order to obtain an even smaller rms-deviation, we fitted allβ-strands of the central β-barrel, giving an rms-deviation of 0.52 Å.When the C-terminal strands and certain diverging residues were nottaken into account, an rms-deviation as low as 0.37 Å was obtained. Thehigh structural resemblance of the central β-barrel of both 1FOR and1NCA ensures us that we have correctly positioned the two domainsrelative to each other.

In the next step, only the V_(K) fragment of 1FOR and the V_(H) of 1NCAwere retained and CDR-loops of 1FOR and 1NCA were adopted withoutfurther modeling.

Modeling of the D9D10 Sequence Onto the Constructed Framework

When the sequences of D9D10 were compared with those of 1FOR-V_(K) and1NCA-V_(H), 21 and 20 mutations were necessary to mutate 1FOR and 1NCAinto D9D10, respectively. These mutations were done simultaneously usingthe Dead-End Elimination method (Desmet et al., 1992) which found theglobally best conformation for all 41 mutations. For both V_(K) andV_(H), the mutations could be done without inducing sterical orenergetical conflicts. As a consequence, we have obtained a veryreliable 3D-model for the variable domains of D9D10 (except for CDR-H3).

PART 2: Humanization of D9D10

Identification of Residues to be Humanized

In order to identify typical D9D10 ‘murine’ residues, V_(K) and V_(H)sequences were again subjected to a BLASTP-search, but this time theentire ‘non-redundant Genbank’ database (PDB+SwissProt+SPupdate+PIR) wassearched for similar sequences. Out of the resulting matches, only humanand humanized sequences were retained and aligned with D9D10.

The alignment revealed several systematic differences in sequencebetween the murine D9D10 molecule and the best matching human V_(K) andV_(H) fragments. From this comparison, we have derived a consensus listof human residues.

Each of these residues was then placed onto the D9D10 model and thefollowing properties were examined: (i) the compatibility with theframework and with neighboring residues, (ii) the solvent accessibilityand (iii) the proximity to the CDR-loops. In general, only D9D10residues which were not found in any human sequence, which werestructurally compatible with the D9D10 framework (and CDR's), and whichwere clearly solvent exposed, were selected for humanization.

For the V_(K) domain we proposed 8 mutations, which were spatiallyclustered into 2 surface patches of 3 residues each plus two isolatedresidues. For the V_(H) domain we pinpointed 9 residues to be humanized.The latter residues formed a surface cluster of 5 residues, one of 2residues and 2 additional isolated residues. For neither of the twodomains, buried residues were retained in the mutation list. The reasonfor this is that we explicitly wanted to preserve the D9D10 frameworkstructure and, also, that buried residues are not ‘visible’ to theimmune system anyway.

Finally, the side-chain conformation of the 8+9 mutations was modeledusing the Dead-End Elimination algorithm. We found that all mutationswere energetically favorable. This strengthened the hypothesis that thehumanization procedure would not affect the antigen binding propertiesof D9D10.

1.2. Construction, Expression, Purification and Evaluation of HumanizedscFvD9D10

Eight substitutions in V_(H)D9D10 and 9 in V_(L)D9D10 had to be carriedout as shown in FIG. 2. Since the different mutations were spread amongthe whole V_(H) and V_(L) sequences, it was decided to assemble thewhole V_(H) and V_(L) sequences out of synthetic oligonucleotides,hereby including all necessary substitutions during the oligonucleotidesynthesis as an alternative to mutagenesis. During the oligonucleotidesynthesis, non-optimal E.coli codons were substituted for more optimalones coding for the same amino acid. Both V_(H) and V_(L) regions wereassembled separately according to the PCR assembly method described byStemmer et al. (1995). The assembled V_(H) and V_(L) regions were firstsubcloned in pGEM-T vectors (PROMEGA Corp., Madison Wis., US) and theircorrect sequence was confirmed by DNA sequencing. Both humanised regionswere subsequently introduced into the pscFvD9D10H6 expression vector(Froyen et al., 1993). For the assembly of the heavy chain, wesynthesized 18 oligo's, 40 nucleotides in length, which collectivelyencode both strands of the V_(H) region from the AlwNI site to the Stylsite. The plus strand as well as the minus strand consist of 9 oligo'sconfigured in such a way that, upon assembly, complimentary oligo's willoverlap by 20 nucleotides. In these oligo's we included mutations bothleading to “humanised” amino acids at the predetermined sites and to“optimised” E. coli codons. !Oligo? ? ? ? !No.? Oligo Seq.  1s5′-CGCGCAGCCGCTGGATTGTTATTACT (SEQ ID NO 3) CGCTGCCCAACCAG-3′  2as5′-CAGCTGCACCTGGGCCATCGCTGGTT (SEQ ID NO 4) GGGCAGCGAGTAAT-3′  3s5′-CGATGGCCCAGGTGCAGCTGGTGCAG (SEQ ID NO 5) AGCGGTAGCGAACT-3′  4as5′-CGCTCGCACCCGGTTTTTTCAGTTCG (SEQ ID NO 6) CTACCGCTCTGCAC-3′  5s5′-GAAAAAACCGGGTGCGAGCGTTAAGA (SEQ ID NO 7) TCAGCTGCAAAGCG-3′  6as5′-TCGGTGAAGGTATAACCGCTCGCTTT (SEQ ID NO 8) GCAGCTGATCTTAA-3′  7s5′-AGCGGTTATACCTTCACCGATTACGG (SEQ ID NO 9) TATGAACTGGGTTA-3′  8as5′-ACCTTGACCCGGCGCCTGTTTAACCC (SEQ ID NO 10) AGTTCATACCGTAA-3′  9s5′-AACAGGCGCCGGGTCAAGGTCTGAAA (SEQ ID NO 11) TGGATGGGTTGGAT-3′ 10as5′-TTTCACCGGTGTAGGTGTTGATCCAA (SEQ ID NO 12) CCCATCCATTTCAG-3′ 11s5′-CAACACCTACACCGGTGAAAGCACCT (SEQ ID NO 13) ACGTTGACGATTTC-3′ 12as5′-CTGAAAACGAAACGACCTTTGAAATC (SEQ ID NO 14) GTCAACGTAGGTGC-3′ 13s5′-AAAGGTCGTTTCGTTTTCAGCCTGGA (SEQ ID NO 15) TACCAGCGTTAGCG-3′ 14as5′-GCTGATCTGCAGGTAGGCCGCGCTAA (SEQ ID NO 16) CGCTGGTATCCAGG-3′ 15s5′-CGGCCTACCTGCAGATCAGCTCTCTG (SEQ ID NO 17) AAAGCGGAAGACAC-3′ 16as5′-GCGCGCAGAAGTAGGTCGCGGTGTCT (SEQ ID NO 18) TCCGCTTTCAGAGA-3′ 17s5′-CGCGACCTACTTCTGCGCGCGTCGCG (SEQ ID NO 19) GTTTCTACGCGATG-3′ 18as5′-GCGCCCTTGGCCCCAGTAATCCATCG (SEQ ID NO 20) CGTAGAAACCGCGAC-3′

After assembly of the 18 40-mer oligonucleotides, the desired fragmentwas PCR amplified using 2 oligonucleotides complementary to the 5′ and3′ end of the fragment respectively. Oligo No. Oligo Seq. 1s5′-CGCGCAGCCGCTGGATTGTTATTAC- (SEQ ID NO 21) 3′ 2as5′-GCGCCCTTGGCCCCAGTAATC-3′ (SEQ ID NO 22)

The resulting 381 bp fragment was cloned into a pGEM-T vector, resultingin PGEM-TV_(H)H and several clones were sequenced. A similar approachwas followed for the light chain. Hereby 14 oligos were synthesized, 248-mers and 12 40-mers, which collectively encode both strands of theV_(L) region from the SacI site to the XhoI site. However, since theSacd site was present exactly on an amino acid substitution site, thisrestriction site could not be retained in the synthetic V_(L) gene. Asan alternative, a Bst1107I site was created which will, after ligationwith the blunted Sacd site, restore the exact V_(L) reading frame. OligoNo. Oligo Seq.  1s 5′-GCGGTATACTGACCCAGAGCCCGGCG (SEQ ID NO 23)ACCATGAGCGCGAGCCCGGGT-3′  2as 5′-CAGGTCAGGGTAACACGTTCACCCGG (SEQ ID NO24) GCTCGCGCTCATGG-3′  3s 5′-GAACGTGTTACCCTGACCTGCAGCGC (SEQ ID NO 25)GAGCTCTAGCATCA-3′  4as 5′-ATGATACCAGAACATATAGCTGATGC (SEQ ID NO 26)TAGAGCTCGCGCTG-3′  5s 5′-GCTATATGTTCTGGTATCATCAGCGT (SEQ ID NO 27)CCGGGTCAGAGCCC-3′  6as 5′-TATCATAGATCAACAGACGCGGGCTC (SEQ ID NO 28)TGACCCGGACGCTG-3′  7s 5′-GCGTCTGTTGATCTATGATACCAGCA (SEQ ID NO 29)ACCTGGCGAGCGGT-3′  8as 5′-CCGCTGAAACGCGCCGGAACACCGCT (SEQ ID NO 30)CGCCAGGTTGCTGG-3′  9s 5′-GTTCCGGCGCGTTTCAGCGGTAGCGG (SEQ ID NO 31)TAGCGGTACCAGCT-3′ 10as 5′-ACGGCTGATGGTCAGGCTATAGCTGG (SEQ ID NO 32)TACCGCTACCGCTA-3′ 11s 5′-ATAGCCTGACCATCAGCCGTATGGAA (SEQ ID NO 33)CCGGAAGATTTCGC-3′ 12as 5′-TCTGATGGCAGAAATAGGTCGCGAAA (SEQ ID NO 34)TCTTCCGGTTCCAT-3′ 13s 5′-GACCTATTTCTGCCATCAGAGCTCTA (SEQ ID NO 35)GCTATCCGTTCACC-3′ 14as 5′-CGCGCTCGAGTTTGGTACCCTGACCG (SEQ ID NO 36)AAGGTGAACGGATAGCTAGAGC-3′

After assembly of the 2 48-mer and 12 40-mer oligonucleotides, thedesired fragment was again PCR amplified using 2 oligonucleotidescomplementary to the 5′ and 3′ end of the fragment respectively. OligoNo. Oligo Seq. 1s 5′-CGCGGTATACTGACCCAGAGC-3′ (SEQ ID NO 37) 2as5′-CGCGCTCGAGTTTGGTACCCTG-3′ (SEQ ID NO 38)

The resulting 316 bp fragment was cloned into a pGEM-T vector, resultingin PGEM-TV_(L)H and several clones were sequenced. The assembly PCRprotocol (Stemmer et al., 1995) consisted of 3 steps: gene assembly,gene amplification and cloning. Since single-stranded ends ofcomplementary DNA fragments were filled-in during the gene assemblyprocess, cycling with Taq DNA polymerase resulted in the formation ofincreasingly larger DNA fragments until the full-length gene wasobtained. It can be noted that DNA ligase has not been used in theprocess. After assembly, the desired fragments were amplified using 5′and 3′ end complementary primers. The resulting fragments weresubsequently cloned into a suitable cloning vector such as pGEM-T,giving PGEM-TV_(L)H and PGEM-TV_(H)H. The final vector,pscFvD9D10V_(Hum), was constructed by ligating a 310 bp Bst1107I/XhoIfragment originating from vector pGEM-TV_(L)H with a 3180 bpSacIblunt/XhoI fragment originating from vector pscFvD9D10H6V_(H)H(=pscFvD9D10H6 in which V_(H) was replaced by the humanized V_(H)obtained from pGEM-TV_(H)H).

Induction of the humanised scFv D9D10 was carried out in E.coli strainJM83. Detection of His6-tagged scFv's on western blot was done with ananti D9D10 rabbit polyclonal antibody and an anti His6 monoclonalantibody (Babco, Richmond, Calif., USA). Compared to the non-humanizedscFvD9D10 (Froyen et al., 1993), the humanized scFvD9D10 was expressedat approximately 3-5 times higher levels (30-40 mg/l). This increase inexpression level can be due to the fact that during assembly thehumanized scFvD9D10 coding sequence was codon-optimised for E. coliexpression. Alternatively, one or several of the humanized amino acidscan have a beneficial effect on the expression level; or the increase inexpression level can be caused by a combination of the two. As with thenon-humanized scFv, most of the expressed protein was still presentintracellularly (70-80%), with 5-10% present in the periplasmic fractionand 10-20% secreted to the medium.

The cells were harvested and lysed in the presence of proteaseinhibitors at 4° C. by the French press (2 passages at 14.000 psi). Thecell lysate was clarified by centrifugation and the supernatant was usedfor purification. The supernatant was loaded on Zn²⁺-IDA Sepharose FFand the resin was washed by applying an imidazole step gradient. Thedifferent pools were analysed by SDS-PAGE under reducing and nonreducing conditions.

The humanized scFv bound and eluted as expected in the 150 mM imidazoleelution pool and SDS-PAGE showed that the recovered scFv was >90% purein a single step. The shift in relative migration under reducingconditions showed that the scFv was purified in an oxidized form.However, in contrast to the mouse scFv, the humanized scFv showed a hightendency for non specific adsorption, because only 40-50% of the initialproduct was recovered after dialysis.

The humanized scFvD9D10 was shown to have the same biological activityas the mouse scFvD9D10 for neutralizing the antiviral activity of humanIFNγ (described in example 7).

Affinity could be calculated for murine and humanized scFv using SurfacePlasmon Resonance(SPR)-analysis with the BIACORE® (Biacore AB, Uppsala,Sweden). This technology permits real-time mass measurements usingsurface plasmon resonance. SPR is an optical phenomenon, seen as a sharpdip in the intensity of light reflected from a thin metal film coatedonto a glass support. The position of this dip depends on theconcentration of solutes close to the metal surface. In general, aprotein (e.g. antibody) is coupled to the dextran layer (covering thegold film) of a sensor chip and solutions containing differentconcentrations of a binding protein (e.g. antigen) are allowed to flowacross the chip. Binding (association and dissociation) is monitoredwith mass sensitive detection.

In order to determine the affinity of the D9D10 derivatives for hIFNγ,BIACORE® experiments were performed in which the murine D9D10 wasimmobilized onto a CM5 sensorchip (Biacore AB). D9D10 was immobilizedusing amine coupling according to the manufacturer's procedure. Todecrease the non specific interaction of human IFNγ with the carboxylicgroups of the dextran layer, the sensorchip was pretreated with 4 cyclesof EDC/NHS—thus reducing the amount of unblocked carboxylic groupsremaining on the sensor surface—before immobilizing D9D10. Then,immobilization of D9D10 was carried out using a continuous flow of 5μl/min on a sensor chip surface initially activated with 17 μl of an0.05M NHS/ 0.2M EDC mixture. 35 μl of typically 3 μg/ml D9D10 wasinjected over the activated surface. Residual unreacted ester groupswere blocked by injecting 17 μl of 0.1M ethanolamine pH 8.5. D9D10 wasimmobilised directly on a CM5 chip at an optimal concentration of 3μg/ml in an acetate buffer pH 5.4 resulting in an immobilization levelof about 600 RU. Most accurate affinity data were obtained by injectinghuman IFNγ and monitoring the subsequent binding of scFvD9D10; thelatter interacting with remaining free epitopes on human IFNγ. On andoff rates were calculated using the BIAevaluation software (Biacore AB).

Results of a typical experiment are shown in FIG. 3 for murine scFvD9D10and in FIG. 4 for humanized scFvD9D10 (These data were generated inseparate experiments). Calculated data were in good agreement. As offrates were hardly detectable for both constructs in most experiments,only on rates are shown for the concentrations tested. These dataclearly indicated that the humanization did not hamper the bindingcharacteristics of the scFv fragment.

Monoclonal antibodies were generated against the humanized scFvD9D10. Afemale BALB/c mouse was immunized (injected intraperitoneally) 3 timeswith humanized scFvD9D10 (i.e., at days 0(50 μg), 32(25 μg) and 56(25μg)). Three months after, a final boost of 25 μg was given. Three daysafter this last injection, spleen cells were retrieved from theimmunized mouse and used for cell fusion. Dissociated splenocytes fromthe immunized mouse were fused with murine myeloma cells SP2/0-Ag14(ATCC, CRL-1581) at a ratio of 10:3 using a polyethylene glycol/DMSOsolution mainly according the procedure-as described by Köhler andMilstein (1975). The fused cells were mixed up and resuspended in DMEMmedium supplemented with hypoxanthine, sodium pyruvate, glutamine, anon-essential amino acid solution, 20% heat-inactivated fetalclone(Hyclone Lab., Utah) and 10% BM-Condimed (Boehringer Mannheim). Thecells were then distributed to 96 well plates to which aminopterin wasadded 24 hours after the cell fusion. Each well contained between 1 to 5growing hybridoma clones at the average. After 8 days supernatant of thewells was collected and screened in an ELISA for binding to humanizedscFvD9D10. The antibodies of the hybridomas thus generated were furthertested for their binding capacity to murine and humanized scFvD9D10 andhuman IgG. Certain monoclonal antibodies derived from this hyper immunemouse did recognize not only humanized scFvD9D10 but also human IgG,indicating the quality of the humanization strategy. Using theantibodies which specifically interact with humanized scFvD9D10 (1D5C5;11E2G6; 10F12A2 available at Innogenetics N.V., Industriepark Zwijnaarde7, Box 4, B-9052 Ghent, Belgium) and do not cross react with the yettested human IgG preparations, an ELISA is generated for detecting andquantifying D9D10 derived constructs in human and primate serum.

Immunization experiments in rabbit and mouse with his-tagged proteinsincluding the humanized scFvD9D10 revealed weak to fairly highimmunogenic responses of the his tail. Consequently, we made a newconstruct and removed the C-terminal hexahistidinetag from the scFvD9D10(humanized scFvD9D10H6⁻). This was done by cutting vectorpscFvD9D10V_(Hum) with XhoI and EcoRI and substituting the His6-tailwith a tandem stop codon and a unique NcoI site for easy identification.This was accomplished using two synthetic oligo's (oligo 1:5′-TCGAGATCAAACGGTAATAGCCATGG-3′ (SEQ ID NO 39); oligo 2:5′-AATTCCATGGCTATTACCGTTTGATC-3′ (SEQ ID NO 40)) which, when annealed,reconstitute the D9D10 V_(L) coding sequence, followed by tandem stopcodons and a unique NcoI site for identification. The annealeddouble-stranded oligo has sticky ends corresponding to a XhoI site atthe 5′ end and EcoRI site at the 3′ end. The oligo was ligated into theXhoI/EcoRI opened pscFvD9D10V_(Hum) vector resulting inpscFvD9D10V_(Hum)[H6⁻]. Expression analysis showed identical expressionlevels and localisation compared to the His6-tagged D9D10 in E. coli.

2. Generation of Humanized, Chimeric D9D10

Two fusion cDNA-genes respectively coding for the heavy and light chainfusion-proteins of the humanized D9D10 whole antibody were constructed.The light chain fusion cDNA consists of the cDNA encoding the mouseD9D10 light chain leader sequence (Ldr), needed for efficient transportof the fusion protein in the host cell, the humanized D9D10 light chainvariable domain cDNA (V_(Lh)), followed by a human immunoglobulinkappa-light chain constant domain (C_(L)).

The heavy chain fusion cDNA consists of the mouse D9D10 light chainleader cDNA-sequence (Ldr), followed by the humanized D9D10 heavy chainvariable domain cDNA (V_(Hh)) and a human IgG1 heavy chain constantdomain (C_(H)═C_(H)1-Hinge-C_(H)2-C_(H)3) cDNA, in which theC1q-complement binding site in the C_(H)2 region, known to inducecomplement activation upon injection of the recombinant antibody, wasmutated (Pro₃₃₁→Ser) (Xu et al., 1994).

PCR Cloning of Human Immunoglobulin Cγ1 and C_(K) cDNA

Total RNA was isolated from human tonsil cells (frozen pellet of ±10⁷cells) following the Chomczynski GuSCN/acid phenol isolation method(Chomczynski and Sacchi, 1987). 140 μg total RNA was obtained. cDNA wasprepared by annealing 700 ng total RNA to 300 ng random hexamers(Pharmacia, Upsala, Sweden) and reverse transcription for 90 min at 42°C. using AMV reverse transcriptase (RT-Stratagene) in a final volume of20 μl (50 mM Tris pH 8.3, 40 mM KCl, 6 mM MgCl2, 5 mM DTT). The reactionwas inactivated by heating at 90° C. for 15 min.

Cloning of the Human C_(K) cDNA:

The cDNA was used as template for PCR amplification of the human C_(K)cDNA using primer sequences based on the Genbank database sequence,accession # V00557 and # J00241. oligo #7061 (Cκ sense primer):              ThrValAla... 5′-TCGAAGCTT AGTACTGTGGCTGCACCATCTGT-3′ (SEQID NO 41)        HindIII ScaI

oligo #7060 (C_(K)  antisense primer):                 CysGluGly...5′-GTCGAATTC TGCGCACTCTCCCCTGTTGAAGC-3′ (SEQ ID NO 42)         EcoRIFspI

PCR amplification using the 7060/7061 primers is expected to yield afragment of 342 basepairs. ScaI/FspI digestion of this fragment shouldyield a blunt fragment starting at the first AA, Thr of C_(K) and endingat the last AA, Cys. A stop codon is not present.

PCR reaction was carried out in a final volume of 50 μl, using 2 μl ofthe RT reaction, 10 pmol of each primer and 5 U of either Taq DNApolymerase (Stratagene, La Jolla, Calif., USA). dNTPs were present at afinal concentration of 200 μM in 1×Taq buffer as provided by thesupplier. Reactions were overlaid with 75 μl paraffin oil. Cyclingconditions were as follows. After an initial denaturation of 5 min at95° C. 40 PCR cycles (1 min 94° C., 1 min at appropriate annealingtemperature of 60° C. and 1 min at 72° C.) were carried out. There was afinal extension phase of 10 min at 72° C. 5 μl amounts of the reactionwere run on agarose gels.

The PCR reaction with the 7060/7061 primer pair yielded a single band of±300 bases, which was purified using the Geneclean™ kit (Bio 101, Vista,Calif., USA), digested with EcoRI/HindIII, phenol:CHCl₃ extracted andligated into EcoRI/HindIII digested pBSK(−) vector (Stratagene). Theligation mix was electroporated into the DH5αF′ bacterial strain.Transformed bacteria were plated onto X-gal/IPTG LB agar plates forblue/white selection of recombinants. Four white colonies were selectedfor further analysis and plasmid DNA was prepared. EcoRI/HindIIIrestriction analysis showed that all 4 C_(K) transformants contained aninsert of the correct length. The 4 inserts were entirely sequenced. Oneclone was completely identical to the database sequence (accession nrsV00557 and J00241). The corresponding plasmid was named pBLSKIGkappaC.

Cloning of the Human Cγ1 Heavy Chain Constant Domain cDNA:

The cDNA was used as template for PCR amplification using primersequences based on the Genbank database sequence: accession # Z17370.oligo #7601 (Cγ1 sense primer; 48-mer, should only be Cγ1 specific)               AlaSerThr... 5′-CTAGAATTCTGCGCATCCACCAAGGGCCCATCGGTCTTCCCCCTGGCA-3′ (SEQ ID NO 43)         EcoRIFspI

oligo #7600 (Cγ1 antisense primer):                LysGlyProSer...5′-GTAAAGCTT GAGCTCTTACCCGGAGACAGGGAGAGG-3′ (SEQ ID NO 44)       HindIII SacI

PCR amplification using the 7601/7600 primer couple is expected to yielda fragment of 1016 basepairs. FspI/SacI cleavage of this fragmentfollowed by removal of the SacI 3′ overhang should yield a bluntfragment starting with the first AA, Ala of Cγ1 and ending with the lastAA, Lys. A stop codon is not included. PCR reactions were carried out ina final volume of 50 μl, using 2 μl of the RT reaction, 10 pmol of eachprimer and 5 U of Taq DNA polymerase (Stratagene). dNTPs were present ata final concentration of 200 μM in 1×Taq buffer as provided by thesupplier. Reactions were overlaid with 75 μl paraffin oil. Cyclingconditions were as follows: after an initial denaturation of 5 min at95° C. 40 PCR cycles (1 min 94° C., 1 min at appropriate annealing temp.55° C. and 1 min at 72° C.) were carried out. There was a finalextension phase of 10 min at 72° C. 10 μl amounts of the reaction wererun on agarose gels. A single band of around 1 kb was obtained. The 1 kbband, obtained with the 7601/7600 primer pair, was purified using theQiaquick™-kit (Qiagen, Hilden, Germany) and ligated into pGEM-T-vector.The ligation mix was transformed into the DH5αF′ bacterial strain.Transformed bacteria were plated onto X-gal/IPTG LB agar plates forblue/ white selection of recombinants.

Eight white colonies were selected for further analysis and plasmid DNAwas prepared. Restriction analysis with BstXI (=specific for IgG-1;absent in IgG-2) showed that 6 transformants contained an Cγ1 insert ofthe correct length. One clone was entirely sequenced and was shown to beidentical to the database sequence, except for 3 codon switches, whichcorrespond to a described allotypic variant Gm(−1,4) of the human IgG1(lys214->arg214, asp356->glu356 and leu358->met358 respectively). Sincethe Gm(−1) (“nonmarker”), glu356/met358, also occurs on Cγ2, this markerwill likely not be immunogenic when introduced in humans. The clonedsequence also contained two silent base switches in comparison to thedatabase sequence Z17370. The final construct was named pGEMThIGG1c.

The C1q-complement binding site present in the C_(H)2 region of thehuman IgG1, known to induce complement activation upon injection of therecombinant antibody (Xu et al., 1994), was later mutated (Pro₃₃₁→Ser)as described further during the assembly of the humanized D9D10 fusioncDNA.

Construction of Fusion cDNAs

In order to assemble the light- and heavy chain fusion genes, severalintermediate cloning constructs, generated by PCR-assembly andamplification, were needed.

Assembly of the Light Chain Fusion cDNA

The mouse D9D10 V_(K) leader sequence cDNA was cloned by PCR-assembly(Stemmer et al., 1995) of four partially overlapping syntheticoligonucleotides [IG8180, IG8179,IG8178 and IG8176] of each 40 bps, andsubsequent PCR-amplification with two specific outside primers [IG 8175and 8174]. The resulting 100 bp PCR fragment I, named Ldr, consist of a5′ untranslated region of 20 bp, including an XbaI cloning site, and thecDNA encoding the complete D9D10 V_(K) leader peptide (20 AA) and 20 bpof the humanized D9D10 light chain variable domain cDNA encoding thefirst 6 AA. Sense strand oligos:                  XbaI IG81805′-GTCCCCCGGGTACCTCTAGAATGGATTTTCAAGTGCAGAT-3′ (SEQ ID NO 45) IG81795′-TTTCAGCTTCCTGCTAATCAGTGCCTCAGTCATACTCTCG-3′ (SEQ ID NO 46)

Antisense strand oligos: IG81785′-CTCTGGGTCAGCTCGATGTCCGAGAGTATGACTGAGGCAC-3′ (SEQ ID NO 47) IG81765′-TGATTAGCAGGAAGCTGAAAATCTGCACTTGAAAATCCAT-3′ (SEQ ID NO 48) PCRamplification primers:                   XbaI IG8175 (sense)5′-GTCCCCCGGGTACCTCTAGAATG-3′ (SEQ ID NO 49) IG8174 (antisense)5′-CTCTGGGTCAGCTCGATGTCC-3′ (SEQ ID NO 50) IG8175→      IG8180                 IG8179 -------------------------------------                  IG8176         IG8178            ----------------- -----------------                                        IG8174

The humanised light chain variable domain as present in pGEM-T-V_(L)H,described earlier, was PCR-amplified using primers [IG8172 and IG8171]designed to produce PCR fragment II containing the complete variabledomain cDNA with exception of the last 3 amino acids (IKR), and flankedat the 3′-terminus by an XhoI-cloning site. IG8172(sense)5′-GACATCGAGCTGACCCAGAGCCCGGCG-3′ (SEQ ID NO 51)         XhoIIG8171(antisense) 5′-CGCGCTCGAGTTTGGTACCCTG-3′ (SEQ ID NO 52)

Fusion of the two DNA fragments PCR-I (Ldr) and PCR-II (V_(Lh)), having20 bp overlap, was performed by overlap PCR using primerset IG8175 andIG8171. The resulting PCR-III fragment was directly cloned in pGEM-Tresulting in the pGEMLdrV_(Lh) plasmid.                   Xba IIG8175(sense) 5′-GTCCCCCGGGTACCTCTAGAATG-3′ (SEQ ID NO 49)         XhoIIG8171(antisense) 5′-CGCGCTCGAGTTTGGTACCCTG-3′ (SEQ ID NO 52)

The human _(K)-light chain constant domain was cloned byPCR-amplification using pBLSKIGkappaC as template with primers IG8170and IG8169. The resulting PCR-IV fragment consists of the cDNA sequenceencoding the last 3 AA of V_(Lh) and the complete human Ckappa constantdomain, followed by a stop codon and an EcoRI cloning site. The PCR-IVDNA was directly cloned in the pGEM-T vector resulting in thePGEM-TC_(L) plasmid.         XhoI IG8170(sense)5′-GCGCCTCGAGATCAAACGGACTGTGGCTGCACCATCTG-3′ (SEQ ID NO 53)        EcoRIIG8169(antisense) 5′-GCCGGAATTCCTAGCACTCTCCCCTGTTGAAG-3′ (SEQ ID NO 54)

Fusion of LdrV_(Lh) and C_(L) cDNA in the pGEM-T backbone was realisedby insertion of the C_(L)-containing XhoI-SpeI fragment, isolated frompGEM-TC_(L) plasmid, in the pGEMLdrV_(Lh) plasmid. The resultingconstruct was named pGEMhD9D10_(L).

Assembly of the Heavy Chain Fusion cDNA

The mouse D9D10 V_(K) leader sequence cDNA was cloned by PCR-assembly(Stemmer et al., 1995) of four partially overlapping syntheticoligonucleotides [IG8180, IG8179,IG8176 and IG8177] of each 40 bps, andsubsequent PCR-amplification with two specific outside primers [IG 8175and 8173]. The resulting 100 bp PCR-V fragment, named Ldr-2, consist ofa 5′ untranslated region of 20 bp, including an XbaI cloning site, andthe cDNA encoding the complete D9D10 V_(K) leader peptide (20 AA) and 20bp of the humanized D9D10 heavy chain variable domain cDNA encoding thefirst 6 AA.

Sense Strand Oligos: Sense strand oligos:                   XbaI IG81805′-GTCCCCCGGGTACCTCTAGAATGGATTTTCAAGTGCAGAT-3′ (SEQ ID NO 45) IG81795′-TTTCAGCTTCCTGCTAATCAGTGCCTCAGTCATACTCTCG-3′ (SEQ ID NO 46) Antisensestrand oligos: IG8177 5′-CTCTGCACCAGCTGCACCTGCGAGAGTATGACTGAGGCAC-3′(SEQ ID NO 55) IG8176 5′-TGATTAGCAGGAAGCTGAAAATCTGCACTTGAAAATCCAT-3′(SEQ ID NO 48)

PCR Amplification Primers: PCR amplification primers:                  XbaI IG8175(sense) 5′-GTCCCCCGGGTACCTCTAGAATG-3′ (SEQID NO 49) IG8173(antisense) 5′-CTCTGCACCAGCTGCACCTGC-3′ (SEQ ID NO 56)IG8175→       IG8180                IG8179 ------------------------------------                 IG8176         IG8177           ----------------- -----------------                                       IG8173

The humanised variable heavy chain domain as present in pGEM-T-V_(H)H,described earlier, was PCR-amplified using primers (IG8168 and IG8167)designed to produce PCR-VI fragment containing the complete variabledomain cDNA, and flanked at the 3′-terminus by an XhoI-cloning site.IG8168(sense) 5′-CAGGTGCAGCTGGTGCAGAGCGGTAG-3′ (SEQ ID NO 57)          XhoI IG8167(antisense)5′-CGCCGGCTCGAGACGGTGACCGTGGTCCCTTGGCCCCAGTAATCC-3′ (SEQ ID NO 58)

Fusion of Ldr-2 and V_(Hh) was performed by overlap PCR on a mixture ofPCR-V and PCR-VI using sense primer IG 8175 and an antisense primer IG8166, resulted in a PCR fragment (LdrV_(Hh)) which was directly clonedin a pGEM-T vector, resulting in pGEMLdrV_(Hh).                   XbaIIG8175(sense) 5′-GTCCCCCGGGTACCTCTAGAATG-3′ (SEQ ID NO 49)          XhoI IG8166(antisense) 5′-CGCCGGCTCGAGACGGTGACC-3′ (SEQ ID NO59)

The human heavy chain constant domain cDNA was produced by PCRamplification on pGEMThIGG1c as template, using sense primer IG 8165,designed to introduce a XhoI restriction site and antisense primer IG8164 that added an extra leucine to the C_(H) sequence and introduced aSTOP codon followed by an EcoRI cloning site. The introduction of acodon for a leucine provided, together with the codon for a lysine(normally the last amino acid), a HindIII restriction site. This HindIIIsite was used to insert a scFv-module (cfr MoTAbII expression plasmids,see below). The resulting fragment PCR-VII was inserted in the pGEM-Tvector resulting in plasmid PGEM-TC_(H).                XhoIIG8165(sense) 5′-GCCGCTCGAGCGCATCCACCAAGGGC-3′ (SEQ ID NO 60)              EcoRI    HindIII IG8164(antisense)5′-GCCGGAATTCGCTAAAGCTTACCCGGAGACAGGGAGAGG-3′ (SEQ ID NO 61)

The amino acid Pro at position 331 in the C_(H)2 domain of both IgG1 andIgG4 immunoglobulins is described to contribute to their differentialability to bind and activate complement (Xu et al., 1994). The Pro331-codon CCC was therefore mutated to a Ser331-codon, TCC. Two specificprimers IG 8460 and IG8459 were designed, to introduce this mutation byPCR mutagenesis.

Two separate PCR-amplifications were performed on PGEM-T-C_(H) astemplate using (1) primers IG2617, matching with the T7-promoter regionin pGEM-T and IG8460, resulting in a 733 bp PCR-VIII fragment, and (2)primers IG 8459 and IG3899, matching the SP6-promoter in pGEM-T,resulting in a 473 bp PCR-IX fragment. Overlap PCR was subsequentlyperformed on a mixture of PCR-VIII and PCR-IX, using again the primersIG2617 and IG3899, resulting in a 1178 bp PCR-X fragment. The amplifiedPCR-X fragment was eventually inserted as an XhoI-SpeI fragment (1018bp) in the pGEMLdrV_(Hh) plasmid. The resulting pGEMhD9D10_(H) plasmidcontains the complete coding sequence of the humanized D9D10 heavy chainfusion protein. IG8459(sense) 5′-GCCCTCCCAGCCTCCATCGAGAAAAC-3′ (SEQ IDNO 62)                Ser₃₃₁ IG8460(antisense)5′-GTTTTCTCGATGGAGGCTGGGAGGGC-3′ (SEQ ID NO 63)                   Ser₃₃₁IG2617(sense-T7) 5′-TAATACGACTCACTA-3′ (SEQ ID NO 64)IG3899(antisense-SP6) 5′-ATTTAGGTGACACTATAG-3′ (SEQ ID NO 65)*Construction of mammalian expression plasmids

Successful high level expression of recombinant immunoglobulins has beenreported in both lymphoid and non-lymphoid mammalian cell lines.Basically an expression plasmid(s), containing the immunoglobulin genescoding for respectively heavy and light chain proteins undertranscriptional control of a promoter/enhancer unit recognized inmammalian cells, is introduced in the chosen host cells together with(as one plasmid or on separate plasmids) a drug-resistance geneexpression unit by classical cell transfection techniques. Cells thathave randomly integrated the foreign expression units in their cellgenome are intially selected for their drug-resistant phenotype andsecondly for high level, stable expression of the protein of interest,the immunoglobulin. After gene integration, an increase in theimmunoglobulin expression level can be obtained by coamplification ofthe genes through further selection of isolated recombinant cell linesfor increased resistance to the drug resistance marker.

One possible example of a successful strategy for mammalian cellexpression is the glutamine synthetase based selection/amplificationmethod shown to result in high level production of mammalian proteins indifferent cell types including Chinese hamster ovary cells (CHO)(Cockett et al., 1990) and myeloma cells, Ns0 (Bebbington et al., 1992).The use of the system is covered by patents WO87/04462 and WO89/10404(Lonza Biologicals, Slough, UK).

Following the GS-expression method, the fusion genes coding forrespectively the heavy- and light chain of the recombinantimmunoglobulins were cloned in a mammalian expression plasmid (pEE12 orpEE14) under transcriptional control of the strong Cytomegalovirus majorimmediate early promoter/enhancer (CMV-MIE). This plasmid also carries acloned glutamine synthetase (GS) gene expression element that can act asa dominant selectable marker in a variety of cells. GS indeed providesthe only pathway for synthesis of glutamine using glutamate and ammoniaas substrates. The final fusion product LdrV_(Lh)C_(L) or hD9D10_(L) wasdirectly cloned as an XbaI-EcoRI fragment isolated from the plasmidpGEMhD9D10_(L) in the mammalian expression vectors pEE14 (for CHO) andpEE12 (for Ns0) (Lonza biologicals) under transcriptional control of theCMV promoter, resulting in the plasmids pEE12hD9D10_(L) andpEE14hD9D10_(L).

The cDNA encoding the heavy chain fusion protein LdrV_(Hh)C_(H) orhD9D10_(H) was first transferred from the pGEMhD9D10_(H) construct as anXbaI-EcoRI fragment in the intermediate vector pEE6hCMV-BglII (LonzaBiologicals), also behind the CMV promoter. From the latter constructpEE6hD9D10_(H) a complete mammalian expression casette, consisting ofCMV-promoter followed by the fusion gene and a polyadenylation site,were transferred as an BglII-BamHI DNA fragment in the BamHI openedplasmids pEE12hD9D10_(L) and pEE14hD9D10_(L) expression plasmids alreadyavailable. The final expression plasmids, named pEE12hD9D10 andpEE14hD9D10 then consists of the pEE-backbone plasmid containing theGS-selection unit, carrying the light chain fusion gene expressioncasette followed by a comparable heavy chain fusion gene expressioncasette.

The approach of assembling a single expression plasmid containingseparate transcription units for both heavy and light chains and theselectable marker is advised in order to ensure coamplification with themarker gene.

A schematic representation of both plasmids is given in FIGS. 5 and 6.

The cDNA sequence encoding the complete humanized D9D10 heavy chainfusion protein is given in FIG. 7. (SEQ ID NO 66)

The cDNA sequence encoding the humanized D9D10 light chain fusionprotein is given in FIG. 8. (SEQ ID NO 68)

The amino acid sequence of the humanized D9D10 heavy chain fusionprotein is given in FIG. 9. (SEQ ID NO 67)

The aminoacid sequence of the humanized D9D10 light chain fusion proteinis given in FIG. 10. (SEQ ID NO 69)

Small Scale Expression of Humanized D9D10 Chimeric Antibody in COS Cells

A quick way to determine the feasibility of expressing a recombinantprotein in mammalian cells and to evaluate its functionality istransient expression of the product in COS cells (Gluzmann, 1981). COScells are Simian Virus 40 (SV40)-permissive CV1 cells (African monkeykidney) stably transformed with an origin-defective SV40 genome, therebyconstitutively producing the SV40 T-antigen. In SV40-permissive cells,T-antigen initiates high copy number transient episomal replication ofany DNA-vector that contains the SV40 origin of DNA replication. Boththe pEE12 and pEE14 expression vectors contain an SV40 origin ofreplication in the SV40 early promoter region controlling theGS-selection gene, and thus permits efficient transient expression inCOS cells.

Small amounts of functionally active antibody were made by transientexpression in COS cells. COS7 cells (ATCC CRL 1651) were routinelycultured in DMEM supplemented with 0.03% glutamine and 10% fetal calfserum. For preparative scale transfection, an optimizedDEAE-transfection protocol (McCutchan, 1968) was used. Alternatively,other well known transfection methods such as Ca-phosphateprecipitation, electroporation, liposome-based transfection can be used.Briefly, exponentially growing COS7 cells were seeded in cell factories(Nunc, Rochester, N.Y., USA) at 3.5 10⁴ cells/cm². about 18 h beforetransfection, after which the cells were washed twice with MEM-Hepes pH7.1 (Gibco, Rockville, Md., USA) and allowed to cool to benchtemperature. 0.5 μg/cm² cell surface of high quality plasmid DNA(CsCl-density purification) of the mammalian expression plasmidspEE12hD9D10 and pEE14hD9D10 was ethanol precipitated, redissolved in 25μl/cm² MEM-Hepes pH 7.1 and slowly added to the same volume of 2 mg/mlDEAE-dextran MW 500.000 (Pharmacia) in MEM-Hepes pH 7.1. TheDNA-DEAE-dextran precipitate (50 μl/cm²) was allowed to form for 20-25min, put on the cells for 25 min and removed to be stored at −20° C.(the same precipitate can be reused in a second transfection experimentwith the same efficiency).

The cells were incubated during the next 3.5 hours in DMEM growth medium(Gibco) containing 0.1 mM chloroquine (Sigma) (0.3 ml /cm²) in aCO₂-incubator at 37° C., then washed two times with growth medium andfurther incubated for 18 hrs in complete culture medium enriched with0.1 mM sodium butyrate (Sigma) at 37° C. (0.3 ml/cm²). The next day thecells were washed twice with serum free DMEM medium supplemented with0.03% glutamine (Merck) and then incubated for 48 h (determined inanalytical scale experiments as the optimal harvest time) in 150 μl/cm²cell surface of the same medium at 37° C., after which conditionedmedium was harvested and stored at −70° C. until purification. Asnegative control COS cells were also transfected with the emptyexpression vectors pEE12 and pEE14.

Quality control of the crude CM was performed by IFNγ-binding assay inELISA format, by SPR-analysis and by measuring the inhibition of IFNγmediated MHC class II-induction.

Human Interferony-Coating Elisa

96 well ELISA culture plates (Nunc 469914) were coated with 100 ng/wellhIFNγ (Genzyme 80-3348-01, 1 mg/ml) diluted in 50 mM TrisHCl pH8.5, 150mM NaCl, by 18 h incubation at 4° C. Blocking of nonspecific binding wasperformed in PBS/0.1% caseine (200 μl/well, 1 h, 37° C.). All washingsteps were performed with PBS/0.05% Tween-20 (3×200 μl/well). Purifiedmouse-human chimeric D9D10 whole antibody (EP 0 528 469 to Billiau andFroyen), produced by transient expression in COS cells, was used aspositive control (concentration range 500 ng/well to 4 ng/well, ½dilution series prepared in the sample diluent, 100 μl/well). Sampleswere diluted in a ½ dilution series in PBS/0.1% caseine, and incubatedfor 2 h at 37° C. Detection was performed using an alkaline-phophataseconjugated goat-anti-human IgG_(H+L) (PromegaS3821), diluted 1/2000 inPBS/0.05% caseine, incubated for 2 h at 37° C. AP-substrate(SigmaN-2765) was used at a concentration of 1 mg/ml in 100 mM TrisClpH9.5, 100 mM NaCl, 5 mM MgCl₂. Plates were analysed at 405/595 nm afterresp. 15 and 30 min incubation at 37° C.

Results are shown in FIG. 11: humanized D9D10 clearly interacts withhuman IFNγ coated onto the wells.

SPR Analysis

A comparable set up was used as described for the evaluation of themurine and humanized scFvD9D10 derivatives. Briefly, murine D9D10 wasimmobilized directly onto a B1 sensorchip (BIACORE AB)—containing lesscarboxylic groups and for which as such no pretreatment is necessary—ata concentration of 10 μg/ml D9D10 in an acetate buffer pH 4.8 usingamine coupling. A fixed concentration of 8 μg/ml human IFNγ was added,followed by the injection of either murine D9D10 (10 μg/ml; positivecontrol) or crude COS supernatant containing humanized D9D10. Resultsare shown in FIG. 12. These data clearly illustrate the presence ofactive, IFNγ binding molecules in the COS supernatant. As no exactconcentrations were determined of the humanized D9D10, no affinity datawere calculated.

Inhibition of MHC Class II-induction See Example 8.1.

Purification of Humanized D9D10

Humanized D9D10 was purified using classical protein A chromatography(Perry and Kirby, 1990; Page and Thorpe, 1996). Quality control of thepurified antibody construct was performed by Western Blot (classicaltechnology) and ELISA. The latter is done as described above and resultsare shown in FIG. 13. From these results it is clear that purified,humanized D9D10 is specifically interacting with IFNγ coated onto thewells.

Generation of Stable Mammalian Expression Cell Lines

For generation of stable mammalian expression cell line, two host celllines Ns0 (Galfre and Milstein, 1981; ECACC 85110503) and CHO-K1 (ATCCCCL61) were used.

The glutamine-dependent NS0 cells were routinely cultured in Lonza DME(JRH 51435)/200 mMglutamine/10% FCS. High quality plasmid DNApEE12hD9D10, prepared by CsCl-density purification, and linearized bySalI digestion, was used for transfection of the NS0 cells byelectroporation (40 μg DNA/10⁷ cells). Transfected cells were thenselected for the glutamine-independent phenotype by gradual reducing theglutamine concentration. Selection was performed in Lonza DME(JRH51435)/GS supplement (JRH58672)/10% dialysed FCS. Individual NSOclones were isolated after ±2 weeks of selection. The clones wereanalysed for recombinant antibody production and secretion by testingthe cell conditioned medium in the IFNγ-coating ELISA described earlier.

Several positive cell lines were selected for subsequent vectoramplification by growth in the presence of the GS-inhibitor MSX(methionine sulfoximine), resulting in increased humanized D9D10antibody expression levels.

Large scale production of the recombinant antibody using high expressingNS0 recombinant cell lines is done in bioreactor systems (e.g. hollowfibre systems)

CHO-K1 cells were routinely cultured in GMEM-S (JRH51492)/200 mMglutamine/10% FCS. High quality plasmid DNA pEE14hD9D10, prepared byCsCl-density purification, was directly used for transfection of CHO-K1cells by Ca²⁺-phosphate transfection techniques (12 μg/1.15 10⁶ cellsseeded 18 h before transfection on T-flasks). Selective medium,GMEM-S(JR51492)/GS supplement (JRH58672)/10% dialysed FCS/25 μM MSX wasadded to the cells 24 h post-transfection. Individual clones could beisolated ±2 weeks after transfection. Selected clones were analysed forrecombinant antibody expression and secretion by testing the cellconditioned medium in the IFNγ-coating Elisa described earlier. Severalpositive cell lines were selected for subsequent vector amplification bygrowth in the presence of increased concentrations of the GS-inhibitorMSX, resulting in increased antibody expression levels.

Large scale production of the recombinant antibody using high expressingCHO-K1 recombinant cell lines is done in bioreactor systems (e.g. hollowfibre or ceramic core systems).

3. Generation of Humanized Sheep Anti-IFNγ Antibodies

Sheep antibodies were generated by immunizing sheeps according tostandard immunization protocols. Briefly, sheeps were injectedintradermally on multiple sites with the antigen (recombinant humanIFNγ(procaryotic origin)) for several times over a timeframe of severalmonths (day 0, 14, 28, 56, extra injections on a monthly basis). Serumis tested for its antiviral activity and its affinity (using SPRanalysis).

As elution conditions necessary to elute an antigen from its antibodyreflect the affinity of the antibody (McCloskey et al., 1997),experiments are performed in which the elution conditions of the sheepantibodies for human IFNγ were compared with those of the scFvD9D10antibody.

Sheep monoclonal antibodies are generated by fusing B-lymphocytesisolated from peripheral blood with murine Sp2/0 myeloma cells accordingto the protocol as described in example 1. The affinity of theantibodies for human IFNγ is determined by SPR analysis as described inexample 1.

4. Generation of Anti-IFNγ Tetravalent Antibody Constructs

4.1. Generation of MoTAb I

The MoTAb I (Monospecific Tetravalent Antibody) molecule is defined as amolecule which consists of 4 identical scFv molecules (e.g. humanizedD9D10 scFv's) in the format of a homodimer of two identical molecules,each containing two scFv's. Both scFv's are linked together using adimerisation domain, which drives the homodimerisation of the molecule(see FIG. 1). Comparable structures have already been described (Pack etal., 1995, Plückthun & Pack, 1997).

The humanized D9D10 scFv was used as a building block to generate theMoTAbI molecule using standard recombinant DNA techniques. A singleMoTAb subunit started with a humanized D9D10 scFv followed by adimerisation domain flanked by flexible linkers. The dimerisation domainwas in turn linked C-terminally to a second D9D10 scFv. Finally adetection and purification tag was added to the extreme C-terminus ofthe molecule. However, in order to circumvent possible immunologicalreactions against the tag, MoTAb I was also produced in an untaggedversion. The sequence coding for the dimerisation domain and theflanking linkers were made synthetically using the method described byStemmer et al. (1995). This synthetic domain was subsequently linked toboth D9D10 scFv's. As linkers between the dimerisation domain and thescFv's, we have used the flexible and proteolysis-resistant truncatedhuman IgG3 upper hinge region (Pack & Plückthun, 1992). As dimerisationdomain we used either the helix-turn-helix motif described by Pack etal. (1993) or the leucine-zipper dimerisation domain originating fromthe human JEM-1 protein as described by Duprez et al. (1997).Optionally, an additional cysteine residue is inserted next to thedimerisation domain to provide extra stability. When applicable, aC-terminal detection and purification tag e.g. a hexahistidine sequence,is used. The sequences were assembled in such a way that functionaldomains were easily replaceable using unique restriction sites presentin the molecule. For the construction of the pGEM-THDH vector, wesynthesized 10 oligo's which collectively encode both strands of the HDHregion (hinge region-dimerization domain-hinge region) flanked by a XhoIand a SpeI restriction site. The plus strand as well as the minus strandconsist of 5 oligo's configured in such a way that, upon assembly,complimentary oligo's will overlap by 20 nucleotides. In these oligo'sthe codons where optimised for optimal E.coli usage. The resulting 223bp fragment was cloned into a pGEM-T vector and several clones weresequenced.

Assembly Oligonucleotides for the HDH-domain: Oligo No. Oligo Seq. 1s5′-CGCGCTCGAGATCAAACGGACCCCGC (SEQ ID NO 70) TGGGTGATACCACTC-3′ 2as5′-CAGTTCACCTCCGGAGGTATGAGTGG (SEQ ID NO 71) TATCACCCAGCGGG-3′ 3s5′-ATACCTCCGGAGGTGAACTGGAAGAG (SEQ ID NO 72) CTGTTGAAACATCT-3′ 4as5′-GACCTTTCAGCAGTTCTTTCAGATGT (SEQ ID NO 73) TTCAACAGCTCTTC-3′ 5s5′-GAAAGAACTGCTGAAAGGTCCGCGGA (SEQ ID NO 74) AAGGTGAACTGGAG-3′ 6as5′-TTCAGGTGCTTCAGCAATTCCTCCAG (SEQ ID NO 75) TTCACCTTTCCGCG-3′ 7s5′-GAATTGCTGAAGCACCTGAAAGAGCT (SEQ ID NO 76) GTTGAAAGGTACCC-3′ 8as5′-ATGGGTAGTATCACCTAGGGGGGTAC (SEQ ID NO 77) CTTTCAACAGCTCT-3′ 9s5′-CCCTAGGTGATACTACCCATACCAGC (SEQ ID NO 78) GGTCAGGTGCAACT-3′ 10as5′-CGCGGAATTCGCGTTCGCGACTAGTT (SEQ ID NO 79) GCACCTGACCGCTGGT-3′

Amplification Oligonucleotides for the HDH-domain: Oligo No. Oligo Seq.1s 5′-CGCGGTATACTGACCCAGAGC-3′ (SEQ ID NO 80) 2as5′-CGCGCTCGAGTTTGGTACCCTG-3′ (SEQ ID NO 81)

The MoTAbI expressionplasmid was constructed as followed: The scFvD9D10coding sequence was amplified by PCR using the pscFvD9D10V_(Hum) plasmidas a template. The sense primer used in this amplification carried aunique SpeI restriction site in such a way that the resulting scFvD9D10sequence could be fused in-frame at the C-terminus of the dimerisationdomain. sense primer: 5′-CGCGACTAGTGCAGAGCGGTAGCGAACTG-3′ (SEQ ID NO 82)antisense primer: 5′-GCCAGTGAATTCTATTAGTGGTGATG-3′ (SEQ ID NO 83)

The resulting PCR fragment was inserted into the pGEM-T vector andverified by DNA sequence analysis. The resulting plasmid was namedpGEM-TscFvD9D10 f s/e. Subsequently, the MoTABI expressionplamid wasassembled in a three-point ligation using following fragments: TheN-terminal scFvD9D10 originating from vector pscFvD9D10V_(Hum) as aXhoI/BcoRI fragment. This fragment also carried the antibioticresistance gene (Amp), the origin of replication and the expression- andsecretion signals. A second fragment, originating from pGEM-THDH cutwith XhoI and SpeI, carried the helix-turn-helix dimerisation domainalready described previously flanked by human IgG3 upper hinge regions.Finally, a third fragment, originating from the SpeI/EcoRI cutpGEM-TscFvD9D10 f s/e plasmid, carried the C-terminal scFvD9D10 with thehexahistidine tag. The final expressionplasmid was named pMoTAbIH6 (FIG.14) and carried the MoTAbI molecule under control of the lac promotorand the pelB signal sequence as the secretion signal (FIGS. 15 and 16).(SEQ ID NO 84 and 85)

To reduce immunogenicity, the hexahistidine sequence was removed usingsynthetic oligo's in a similar way as described previously for thehumanized scFvD9D10, resulting in MoTabI. The MoTAb I expression plasmidwas introduced into a suitable E.coli expression strain, e.g . JM83 andBL21. Good expressionlevels could be obtained in both strains. Detectionof the MoTabI molecule (60 kDa) on western blot was done with an antiD9D10 rabbit polyclonal antibody and/or an anti His6 monoclonal antibody(Babco). However, only a minor amount of the MoTAbI molecule was presentin a soluble form in the bacterial periplasm. The majority of the MoTAbImolecule was not able to traverse the bacterial membrane and was presentas cytoplasmic inclusion bodies. This was confirmed by N-terminal aminoacid sequencing which revealed still the presence of the pelB signalsequence on the molecule. The functionality of the minor amount ofsecreted MoTAbI could however be confirmed using an ELISA. In thisELISA, recombinant human IFNγ was coated onto a polystyreneplate andincubated with periplasmic fractions originating from E.coli cellsexpressing the MoTAbI molecule. Bound MoTAbI molecules where thendetected using a rabbit polyclonal serum generated against the D9D10scFv followed by a peroxidase labeled goat anti rabbit secondary serum.

Since most MoTAbI molecules were present in cytoplasmic inclusionbodies, the molecules were purified from this fraction under denaturingconditions followed by refolding to functional molecules. However, sincethe MoTAbI molecule has the pelB signal sequence still attached, a newcytoplasmic expressionplasmid was constructed. In thisexpressionplasmid, MoTAbI expression is under control of the strongleftward promotor of phage lambda (P_(L)). Since no secretion to theperiplasmic space is necessary, the MoTAbI coding sequence was fuseddirectly to an ATG startcodon. This was accomplished by isolating theMoTAbI coding sequence lacking the pelB signal sequence by PCR from thepMoTAbI expressionplasmid and recloning it into the EcoRV opened pBSK(+)vector (Stratagene). A SapI restriction site giving access to the firstmature codon was hereby generated. After DNA sequence verification theMoTAbI coding sequence was inserted as a SapI blunt/SalI fragment intothe NcoI blunt/ SalI cut pIGRI2 vector. *pIGRI2 expressionvectornucleotide sequence    1TTCCGGGGATCTCTCACCTACCAAACAATGCCCCCCTGCAAAAAATAAAT (SEQ ID NO 86)   51TCATATAAAAAACATACAGATAACCATCTGCGGTGATAAATTATCTCTGG  101CGGTGTTGACATAAATACCACTGGCGGTGATACTGAGCACATCAGCAGGA  151CGCACTGACCACCATGAAGGTGACGCTCTTAAAAATTAAGCCCTGAAGAA  201GGGCAGGGGTACCAGGAGGTTTAAATCATGGTAAGATCAAGTAGTCAAAA  251TTCGAGTGACAAGCCTGTAGCCCACGTCGTAGCAAACCACCAAGTGGAGG  301AGCAGTAACCATGGTTACTGGAGAAGGGGGACCAACTCAGCGCTGAGGTC  351AATCTGCCCAAGTCTAGAGTCGACCTGCAGCCCAAGCTTGGCTGTTTTGG  401CGGATGAGAGAAGATTTTCAGCCTGATACAGATTAAATCAGAACGCAGAA  451GCGGTCTGATAAAACAGAATTTGCCTGGCGGCAGTAGCGCGGTGGTCCCA  501CCTGACCCCATGCCGAACTCAGAAGTGAAACGCCGTAGCGCCGATGGTAG  551TGTGGGGTCTCCCCATGCGAGAGTAGGGAACTGCCAGGCATCAAATAAAA  601CGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTC  651GGTGAACGCTCTCCTGAGTAGGACAAATCCGCCGGGAGCGGATTTGAACG  701TTGCGAAGCAACGGCCCGGAGGGTGGCGGGCAGGACGCCCGCCATAAACT  751GCCAGGCATCAAATTAAGCAGAAGGCCATCCTGACGGATGGCCTTTTTGC  801GTTTCTACAAACTCTTTTGTTTATTTTTCTAAATACATTCAAATATGTAT  851CCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATAAAAGGATCT  901AGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAG  951TTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTC 1001TTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAAC 1051CACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTT 1101TTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCT 1151TCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGC 1201CTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGC 1251GATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAA 1301GGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGG 1351AGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCATTGAGAA 1401AGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGG 1451CAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCT 1501GGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGA 1551TTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAA 1601CGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGT 1651TCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTT 1701GAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTC 1751AGTGAGCGAGGAAGCGGAAGAGCGCTGACTTCCGCGTTTCCAGACTTTAC 1801GAAACACGGAAACCGAAGACCATTCATGTTGTTGCTCAGGTCGCAGACGT 1851TTTGCAGCAGCAGTCGCTTCACGTTCGCTCGCGTATCGGTGATTCATTCT 1901GCTAACCAGTAAGGCAACCCCGCCAGCCTAGCCGGGTCCTCAACGACAGG 1951AGCACGATCATGCGCACCCGTGGCCAGGACCCAACGCTGCCCGAGATGCG 2001CCGCGTGCGGCTGCTGGAGATGGCGGACGCGATGGATATGTTCTGCCAAG 2051GGTTGGTTTGCGCATTCACAGTTCTCCGCAAGAATTGATTGGCTCCAATT 2101CTTGGAGTGGTGAATCCGTTAGCGAGGTGCCGCCGGCTTCCATTCAGGTC 2151GAGGTGGCCCGGCTCCATGCACCGCGACGCAACGCGGGGAGGCAGACAAG 2201GTATAGGGCGGCGCCTACAATCCATGCCAACCCGTTCCATGTGCTCGCCG 2251AGGCGGCATAAATCGCCGTGACGATCAGCGGTCCAGTGATCGAAGTTAGG 2301CTGGTAAGAGCCGCGAGCGATCCTTGAAGCTGTCCCTGATGGTCGTCATC 2351TACCTGCCTGGACAGCATGGCCTGCAACGCGGGCATCCCGATGCCGCCGG 2401AAGCGAGAAGAATCATAATGGGGAAGGCCATCCAGCCTCGCGTCGCGAAC 2451GCCAGCAAGACGTAGCCCAGCGCGTCGGCCGCCATGCCGGCGATAATGGC 2501CTGCTTCTCGCCGAAACGTTTGGTGGCGGGACCAGTGACGAAGGCTTGAG 2551CGAGGGCGTGCAAGATTCCGAATACCGCAAGCGACAGGCCGATCATCGTC 2601GCGCTCCAGCGAAAGCGGTCCTCGCCGAAAATGACCCAGAGCGCTGCCGG 2651CACCTGTCCTACGAGTTGCATGATAAAGAAGACAGTCATAAGTGCGGCGA 2701CGATAGTCATGCCCCGCGCCCACCGGAAGGAGCTGACTGGGTTGAAGGCT 2751CTCAAGGGCATCGGTCGGCGCTCTCCCTTATGCGACTCCTGCATTAGGAA 2801GCAGCCCAGTAGTAGGTTTGAGGCCGUGAGCACCGCCGCCGCAAGGAATG 2851GTGCATGTAAGGAGATGGCGCCCAACAGTCCCCCGGCCACGGGGCCTGCC 2901ACCATACCCACGCCGAAACAAGCGCTCATGAGCCCGAAGTGGCGAGCCCG 2951ATCTTCCCCATCGGTGATGTCGGCGATATAGGCGCCAGCAACCGCACCTG 3001TGGCGCCGGTGATGCCGGCCACGATGCGTCCGGCGTAGAGAATCCACAGG 3051ACGGGTGTGGTCGCCATGATCGCGTAGTCGATAGTGGCTCCAAGTAGCGA 3101AGCGAGCAGGACTGGGCGGCGGCCAAAGCGGTCGGACAGTGCTCCGAGAA 3151CGGGTGCGCATAGAAATTGCATCAACGCATATAGCGCTAGCAGCACGCCA 3201TAGTGACTGGCGATGCTGTCGGAATGGACGATATCCGGCAAGAGGCCCGG 3251CAGTACCGGCATAACCAAGCCTATGCCTACAGCATCCAGGGTGACGGTGC 3301CGAGGATGACGATGAGCGCATTGTTAGATTTCATACACGGTGCCTGACTG 3351CGTTAGCAATTTAACTGTGATAAACTACCGCATTAAAGCTAATCGATGAT 3401AAGCTGTCAAACATGAGAATTAA

The new vector is called pIGRI2MoTAbI. A version lacking thehexahistidine tag as constructed in a similar way starting from theprevious MoTAbI expressionplasmid without hexahistidine tail. The newMoTAbI expressionvectors were subsequently transferred to E.coliexpressionstrains MC1061(pAcI), SG4044(pcI857) and UT5600(pAcI). Asexpected, most of the expressed MoTAbI was present as cytoplasmicinclusionbodies. MoTAbI molecules were purified from cytoplasmicinclusion bodies under denaturing conditions followed by standardrefolding procedures as described by De Bernardez Clark (1998).

4.2. Generation of MoTAb II

The D9D10 MoTAb II is defined as a humanized D9D10 whole antibodymolecule to which a humanized D9D10ScFv sequence was attached at thecarboxyterminus (CH3-domain) of the heavy chain (see FIG. 1). Acomparable type of molecule has already been described in literature(Coloma and Morrison, 1997).

For the expression of the D9D10 MoTAbII protein two fusion genes,respectively coding for heavy and light chain protein of the assembledantibody, were constructed. The heavy chain fusion gene consists of animmunoglobulin leader sequence (D9D10 V_(K) leader cDNA) followed by thehumanized D9D10 heavy chain variable domain cDNA, a human IgG1 heavychain constant domain (C_(H)1-Hinge-C_(H)2-C_(H)3) cDNA, a short G₃Slinker sequence (Coloma and Morrison, 1997) and the humanized D9D10 ScFvsequence. Alternative linker sequences such as the (G₄S)₃ sequence orthe flexible and proteolysis-resistant truncated mouse IgG3 upper hingeregion (Pack & Plückthun, 1992) can be used.

The light chain fusion gene is identical to the humanized D9D10recombinant antibody light chain gene (2) and contains the D9D10 V_(K)leader, the humanized light chain variable domain cDNA and the humanIgG1 constant domain (kappa).

Construction of MoTAb II Heavy Chain cDNA

The basic constructs generated for expression of the humanized D9D10antibody could be used as backbone for the MoTAbII constructs. Asdescribed several intermediate cloning constructs, mainly generated byPCR-assembly and -amplification, eventually resulted in two finalconstructs, named pGEMhD9D10_(L) and pGEMhD9D10_(H). The latter plasmidwas used as acceptorfragment after digestion with HindIII and EcoRI,which eliminates the STOP codon for insertion of a HindIII-EcoRIdonorfragment isolated from a plasmid pGEM-T-D9D10HE, resulting in thein frame fusion of the hD9D10_(H) cDNA to a cDNA sequence encoding theGly₃Ser linker followed by the humanizedScFv-module and a STOP codon.The resulting plasmid was named pGEM-MoTAbII_(H).

pGEM-T-D9D10HE was constructed by PCR amplification usingpScFvD9D10V_(Hum) as template with primers IG8078 and IG8077. Theresulting 755 bp PCR fragment, containing the Gly₃Ser linker followed bythe humanized scFv-module and a STOP codon, was directly cloned in thepGEM-T vector. IG8078 (sense):       HindIII (SEQ ID NO 87)5′-CCCAAGCTTGGCGGAGGCTCACAGGTGCAGCTGGTGCAGAG-3′      EcoRI IG8077(antisense): 5′-CGGAATTCTACCGTTTGATCTCGAGTTTGG-3′ (SEQ ID NO 88)

Construction of Mammalian Expression Plasmids

Expression in mammalian cell lines was performed completely as describedfor the humanized D9D10 antibody (cf example 2). The cDNA encoding theLdrV_(Hh)C_(H)ScFv or MoTAbII_(H) fusion protein was initially insertedin the pEE6hCMV-BglII (Lonza biologicals) intermediate expressionvector, under transcriptional control of the hCMV promoter. This wasperformed by transfer of the EcoRI-XbaI DNA insert from pGEMMoTAbII_(H)into the pEE6hCMV-BglII vector. From the pEE6MoTAbII_(H) plasmid acomplete mammalian expression casette, consisting of CMV-promoterfollowed by the fusion gene and a polyadenylation site, was thentransferred as a BglII/BamHI fragment to the BamHI openedpEE12hD9D10_(L) and pEE14hD9D10_(L) expression plasmids alreadyavailable (construct was earlier described for the humanized D9D10antibody construct in example 2). The final expression plasmids, namedpEE12MoTAbII and pEE14MoTAbII then consisted of the pEE-backbone plasmidcontaining the GS-selection unit, carrying the light chain fusion geneexpression casette followed by a comparable heavy chain fusion geneexpression casette. A schematic representation of both plasmids is givenin FIGS. 17 and 18. The approach of assembling a single expressionplasmid containing separate transcription units for both heavy and lightchains and the selectable marker, is adviced in order to ensurecoamplification with the marker gene. The cDNA sequence encoding thecomplete MoTAbII heavy chain fusion protein is given in FIG. 19 (SEQ IDNO 89). The amino acid sequence of the MoTAbII heavy chain fusionprotein is given in FIG. 20 (SEQ ID NO 90).

Small Scale Expression of D9D10 MoTAbII in COS Cells

Transient expression in COS monkey kidney cells was performed using bothmammalian expression constructs pEE12MoTAbII and pEE14MoTAbII completelyas described for the humanized D9D10 antibody (cf example 2). Qualitycontrol was performed by IFNγ-binding ELISA and SPR-analysis.

ELISA

The same set up was used as described in example 2. Results are shown inFIG. 11. Specific binding to IFNγ is detected. The signal is lower thanthe signal obtained with crude COS supernatant of humanized D9D10.However, no concentrations were determined of MoTAbII.

SPR Analysis

A similar set up was used as described for the evaluation of the murineand humanized scFvD9D10 derivatives. Briefly, murine D9D10 wasimmobilized directly onto a B1 sensorchip at a concentration of 10 μg/mlD9D10 in an acetate buffer pH 4.8 using amine coupling. A fixedconcentration of 8 μg/ml human IFNγ was added, followed by the injectionof either murine D9D10 (10 μg/ml; positive control) or crude COSsupernatant containing MoTAb II. Results are shown in FIG. 21. Thesedata clearly illustrate the presence of active, IFNγ binding moleculesin the COS supernatant. As no exact concentrations were determined ofthe MoTAB II, no affinity data could be calculated.

Inhibition of MHC Class I Induction cf Example 8.1.

Purification

MoTAbII was purified using classical protein A chromatography (Perry andKirby, 1990; Page and Thorpe, 1996). Quality control of the purifiedconstruct was done by Western Blot (classical technology) and ELISA. Thelatter was performed as described in example 2 and results are shown inFIG. 13. From these results we can conclude that MoTAbII is specificallyinteracting with human IFNγ.

Generation of Stable Mammalian Expression Cell Lines

For generation of stable mammalian expression cell line, two host celllines Ns0 (Galfre and Milstein, 1981; ECACC 85110503) and CHO-K1 (ATCCCCL61) were used. Transfection and selection procedures were completelyidentical as described for the humanized D9D10 whole antibody, using theplasmids pEE12MoTAbII for Ns0 and pEE14MoTAbII for CHO-K1. For both NS0and CHO-K1, several MoTAbII producing cell lines (determined inIFNγ-binding ELISA) were initially isolated and used as parental clonesfor further amplification of recombinant protein expression levels asdescribed earlier.

Production of large amounts of the recombinant protein is performed onbioreactor systems optimal for the respective host cells.

5. Generation of Anti-IFNγ Diabodies

Diabodies are dimeric antibody fragments. In each polypeptide, aheavy-chain variable domain (V_(H)) is linked to a light-chain variabledomain (V_(L)) but unlike scFv's, each antigen-binding site is formed bypairing of one V_(H) and one V_(L) domain from two differentpolypeptides. This is achieved by shortening the linker between theV_(H) and V_(L) domains in each molecule (Holliger et al., 1993). Sincediabodies have two antigen-binding sites they can either be monospecificor bispecific. Monospecific bivalent molecules are generated by theshortening the flexible linker sequence of the scFv molecule to betweenfive and ten residues and by cross-pairing 2 scFv molecules withshortened linker. In order to stabilize the molecule, an optionalcysteine residue can be inserted in the linker. As an example for thedifferent steps involved in such a construction we have documented theconstruction of D9D10-derived monospecific, humanized anti-IFNγdiabodies. The 15 residue linker of the His6-tagged, humanized scFvD9D10was replaced by the 5 or 10 residue linker using overlap extension PCR.Shortly, both D9D10 V_(H) and V_(L) coding sequences were PCR amplifiedwhereby the V_(H) antisense primer and the V_(L) sense primer havesequences coding for the 5- or 10-mer linker sequence. The resultingV_(H) and V_(L) PCR fragments were subsequently mixed and a second PCRwith the V_(H) sense and V_(L) antisense primers was performed. Theresulting PCR fragment is cloned into the pBSK(+) plasmid (Stratagene)en verified by DNA sequence analysis (FIGS. 22-25) (SEQ ID NO 91-94).The D9D10 diabody coding sequence was subsequently transferred as a SapIblunt/EcoRI fragment and inserted into the NcoI blunt/EcoRI openedvector pTrc99A (Amann et al., 1988). In this vector, expression of thediabodies is under control of the IPTG inducible Trc promotor. Thediabodies were expressed in E. coli strains HB101 or JM83. Periplasmicfractions were prepared following a modified protocol described by Neuand Heppel (1965). Briefly, cells were harvested by centrifugation andresuspended in ice cold shockbuffer (100 mM Tris-HCl pH 7.4; 20%sucrose; 1 mM EDTA pH8). After incubation on ice during 10 min. withoccasional stirring, the mixture was centrifuged at 10.000 rpm during1,5 min. The supernatans was removed and the pellet was immediatelyresuspended in ice cold distilled water. After incubation on ice during10 min. with occasional stirring, the mixture was centrifuged at 14,000rpm and the obtained supernatans was the soluble periplasmic fraction.The periplasmic fractions were tested for binding to IFNγ usingSPR-analysis. The experimental set up was as described in example 2. Theundiluted samples were injected onto the surface of a B1 sensorchipcoated with murine D9D10 onto which IFNγ was injected. Results obtainedwith L5 D9D10 diabodies are shown in FIG. 26. A clear, specific bindingof the diabodies was detected. Comparable results were obtained with theL10 D9D10 diabody.

The bivalent, monospecific diabody molecules are purified from theperiplasmic extract via IMAC or from periplasmic inclusion bodies usingdenaturing conditions followed by refolding.

Overlap Extension PCR Primers for the L10 D9D10 Diabodies: D9D10V_(H)forward (sense) primer 5′-GGCCGCTCTTCGAAATACCTATTGCCTACGG (SEQ ID NO 95)CAG-3′ D9D10L10V_(H) backward (antisense) primer5′-CTGGGTCAGTACGATGTCAGAGCCACCTCCG (SEQ ID NO 96)CCTGAACCGCCTCCACCTGAGGAGACGGTGACCG TGGTC-3′ D9D10L10V_(L) forward(sense) primer 5′-GTCACCGTCTCCTCAGGTGGAGGCGGTTCAG (SEQ ID NO 97)GCGGAGGTGGCTCTGACATCGTACTGACCCAGAG CC-3′ D9D10V_(L) backward (antisense)primer 5′-GCCAGTGAATTCTATTAGTGGTGATG-3′ (SEQ ID NO 98)

Overlap Extension PCR Primers for the L5 D9D10 Diabodies: D9D10V_(H)forward (sense) primer 5′-GGCCGCTCTTCGAAATACCTATTGCCTACGG (SEQ ID NO 95)CAG-3′ D9D10L5V_(H) backward (antisense) primer5′-CTGGGTCAGTACGATGTCTGAACCGCCTCCA (SEQ ID NO 99)CCTGAGGAGACGGTGACCGTGGTC-3′ D9D10L5V_(L) forward (sense) primer5′-GTCACCGTCTCCTCAGGTGGAGGCGGTTCAG (SEQ ID NO 100)ACATCGTACTGACCCAGAGCC-3′ D9D10V_(L) backward (antisense) primer5′-GCCAGTGAATTCTATTAGTGGTGATG-3′ (SEQ ID NO 98)6. Generation of Anti-IFNγ Triabodies

The construction of triabody molecules was analogous to the schemedescribed above for diabody molecules, except that the (G₄S)₃ linkerbetween the humanized D9D10 VH and VL was completely deleted (FIGS. 27and 28) (SEQ ID NO 101-102) (zero-residue linker or −1-residue linkeraccording to the Kabat numbering (Kortt et al., 1997; Iliades et al.,1997) ). The humanized D9D10 triabody construct is a mono-specificmolecule resulting from the spontaneous association of threezero-residue linker (or −1-residue) D9D10 scFv molecules in thebacterial periplasm. A trimer was formed whereby three pairs of V_(H)and V_(L) domains interact to form three active antigen combining sites.If necessary, in order to drive triabody formation as well as tomaintain stability, we can explore the possibility of introducingadditional association domains or disulfide bridges.

The produced triabodies were tested for IFNγ binding using SPR-analysis.Periplasmic fractions were prepared as described in example 5.SPR-analysis was performed as described in example 5. Results are shownin FIG. 29. A clear, specific binding of the triabody was obtained.

The triabody molecules were purified from the periplasmic extract, madefrom uninduced bacterial cultures, via IMAC and further by gelfiltration or alternatively by purification under denaturing conditionsfrom periplasmic inclusionbodies followed by refolding. The multimericbehaviour of the purified molecules was analysed. The ability of thepurified triabody to bind human interferon y was tested usingSPR-analysis and ELISA experiments as described earlier. For these testswe produced milligram amounts of highly purified material in a suitableE. coli expression system.

Overlap Extension PCR Primers for the L0 D9D10 Triabodies: D9D10V_(H)forward (sense) primer 5′-GGCCGCTCTTCGAAATACCTATTGCCTACGG (SEQ ID NO 95)CAG-3′ D9D10L0V_(H) backward (antisense) primer5′-CTGGGTCAGTACGATGTCTGAGGAGACGGTG (SEQ ID NO 103) ACCGTGGTC-3′D9D10L0V_(L) forward (sense) primer 5′-GTCACCGTCTCCTCAGACATCGTACTGACCC(SEQ ID NO 104) AGAGCC-3′ D9D10V_(L) backward (antisense) primer5′-GCCAGTGAATTCTATTAGTGGTGATG-3′ (SEQ ID NO 98)7. Generation of MoTAb's (and BiTAb's) Originating From Fusion Proteins,From Serum Multisubunit Proteins and From scFv's

The multi subunit (oligomeric) structure of proteins may be exploited toobtain multivalent antibodies, when they are used as fusion partner withscFv antibodies. Either the whole polypetide chain, or the associationsequence domain may be used as fusion partner.

For example, haemoglobin is a tetrameric serum protein, consisting from2 alpha and 2 beta globin subunits. The dimer dissociation constant isestimated to be in the order of 1 nM (Pin et al., 1990). Thetetramer—dimer dissociation constant of haemoglobin in oxy-conformationwas studied by gel filtration on Superose 12 and was calculated to be 1μM (Manning et al, 1996). Although non-covalent associations are knownto be susceptible to equilibrium rules, it has been described that thesubunit interactions are favoured in concentrated protein solutions likeserum and also may be increased by the presence of other stabilisingcompounds (Srere and Mathews, 1990).

Recombinant haemoglobin expression has been extensively investigated asa possible blood substitute in order to circumvent the transmission ofinfectious disease agents during blood transfusion. The alpha- andbeta-globin polypeptides have already been expressed from a singleoperon in E. coli (Hoffman et al., 1990). In this case, the recombinanthaemoglobin was purified from the soluble cytoplasmatic fraction and thetetrameric E. coli product had essentially the same characteristics asthe native protein. Analogous results were obtained when recombinanthaemoglobin was expressed in S. cerevisiae (Pagnier et al., 1992; Mouldet al., 1994; Sutherland-Smith et al., 1998).

Protein engineering strategies (Olson et al., 1997) and chemicalmodification by pegylation (Pettit and Gombotz, 1998) are investigatedto enhance the stability and the circulation half times in vivo. Sofusion of relevant scFv molecules to the respective alpha and betasubunit of human haemoglobin and expression of the fusion proteins froma single operon in either E. coli or S. cerevisiae would yield afunctional tetrameric monospecific (if identical scFv's are used) orbispecific (when different scFv's are used) molecules at high level.

8. Evaluation of Anti-IFNγ Neutralizing Molecules

8.1. Inhibition of MHCII-induction

In the first experiments, the effect of IFNγ on the induction of MHCclass II expression on human keratinocytes was examined. For this,primary human keratinocytes (passage 1) were cultured with twoconcentrations of human IFNγ (100 U/ml and 200 U/ml) during 24 and 48hours. After culture, cells were collected and the expression of MHCclass II antigen on the activated keratinocytes was measured byFACS-scan after staining (30 minutes at 4° C.) of the cells with aPE-labelled anti-MHC-class II mAb. The results showed that restingkeratinocytes do not express MHC class II molecules and that IFNγinduces the expression after 24 hours in a dose-dependent way. Theinduction is still enhanced after 48 hours of culture.

In the next study, the effect of anti-human IFNγ D9D10H3 full sizeantibody or scFvD9D10-cmyc on the IFNγ-induced MHC-Class II expressionon human keratinocytes was examined. In this experiment, human primarykeratinocytes (passage 1) were cultured with human IFNγ (100 U/ml) inthe presence or absence of different concentrations (2-0.5-0.12-0.03)D9D10 Ab or D9D10scFv for 48 hours. IFNγ was preincubated with D9D10H3or scFvD9D10 during 1 hour at 37° C. before adding to the keratinocytes.After culture, cells were collected and the expression of MHC-Class IIon these activated keratinocytes was measured. For this, keratinocyteswere incubated (30 minutes at 4° C.) with a PE-labelled anti-MHC-ClassIImAb (Becton Dickinson), washed twice with PBS and fixed. The MHC-ClassII expression was further analysed on a FACS-scan. The results of theseexperiments are represented in FIG. 30. It is shown that the MHC classII antigen is not expressed on the membrane of resting keratinocytes andthat IFNγ clearly induces this MHC class II expression. This IFNγinduced MHC class II expression is dose dependently inhibited by D9D10H3and to a lesser extent by scFvD9D10. We can conclude that about 4 timesmore scFv (0.12 μg/ml) than full size antibody (0.5 μg/ml) is needed toobtain a 50% inhibition of the IFNγ-induced MHC classII expression onkeratinocytes.

Similar experiments were performed in order to evaluate theneutralization capacity of humanized D9D10 and MoTAbII. Results aresummarized in FIG. 31. Although in this experiment, MHC class IIinduction could be only induced to a lesser extent, both humanized D9D10and MoTAbII clearly inhibit the IFNγ-induction.

8.2. Inhibition of Anti-Viral Activity

For neutralization of the antiviral activity of hIFNγ, serial dilutionsof samples (anti-IFNγ constructs) were prepared in microtiter plates. Toeach well, hIFNγ was added to a final concentration of 5 antiviralprotection Units/ml, as tested on A549 cells. The mixtures wereincubated for 4 h at 37° C. and 25000 A549 cells were added to eachwell. After an incubation period of 24 at 37° C. in a CO₂ incubator, 25μl of 8×10⁵ PFU EMC virus/ml was added to the cultures for at least 24h. As soon as virus-infected control cultures reached 100% celldestruction, a crystal violet staining was performed in order toquantify surviving cells. The neutralization capacity of the anti-IFNγconstructs was defined by the concentration of the construct needed toneutralize 95% of the antiviral activity of 5 U/mi human IFNγ. Theneutralization potency of the scFvD9D10 and the humanized scFvD9D10 wasdetermined and was 1.2 μg/ml and 1.5 μg/ml, respectively.

8.3. Beneficial Effects in Septic Shock in Mice

Septic shock has been demonstrated to be a complex human diseasemanifestation that occurs after the release of lipopolysaccharide (LPS)into the circulation. The subsequent production of high cytokine levelsin the serum are known to play a crucial role in septic shock. Wegenerated data in a mouse model system using an anti-mouse IFNγ calledF3 (Froyen et al., 1995).

The generalized Shwartzman model is a lethal shock syndrome inexperimental animals which is elicited by 2 consecutive injections ofLPS. In the laboratory of prof. Billiau (Rega Institute, CatholicUniversity Leuven, Belgium), such a model was developed in mice (Billiauet al., 1987). At time 0, the mice were injected with 5 μg LPS into thefootpad, followed 24 h later by a second intravenous injection of 100μg. Morbidity and mortality was scored for 5 days. Untreated animalsnormally died within 2 days after the second injection. Mice pretreatedwith the anti-muIFNγ antibody F3 were completely protected against thelethal effect and only showed moderate disease symptoms. This protectioncould be achieved with as little as 2.4 μg F3 given 24 h before thefirst injection. In order to score the severity of the disease, thesymptoms were classified in 5 groups:

-   Score 0: not sick or mild piloerection-   Score 1: piloerection and diarrhoea-   Score 2: hemorhagic conjunctivitis and bleeding at the mouth and    anus-   Score 3: paralysis of the hind legs-   Score 4: death    The highest score that could be obtained is 4. Since the number of    mice in each group was relatively low (5), we established a limit of    the disease score (=2) that had to be reached in the saline group in    order to be a representative experiment.

The schedule we used in order to compare F3 and its scFv in thisShwartzman model was as follows: NMRI mice were given the preparativedose of 5 μg LPS at time 0. At the time points +6 h, +12 h and +23 h themice were injected ip with 190 μg scFvF3 (Froyen et al., 1995) or 30 μgF3. Control animals were given saline at the same time points. Eachgroup consisted of 5 mice. The mice were given a score according to theabove mentioned classification.

In the first experiment, 40% more mice were protected in the scFvF3group when compared with the control group. A second experiment was setup using a slightly adapted protocol: an additional injection was givenat timepoint +3 h. The result of this experiment (shown in table) wassimilar to that of experiment 1 in that 40% more mice survived in thescFvF3 group in comparison with the control group as can be seen in FIG.32. In addition to scFvF3, a Fab antibody fragment of F3 was included inthe second group. All these mice survived the experiment.

The mean disease scores of these experiments, demonstrate a significantdifference for both F3 and the scFv compared to the control group. Themean disease scores of the 5 mice of each group were as follows: SalinescFvF 3F3 FabF3 exp. 1 3.2 1.8 0.0 ND exp. 2 2.6 0.8 0.6 0.68.4. Beneficial Effects During Cachexia in Mice

In a model for cachexia developed at the Rega Institute (Matthys et al.,1991), nude mice were injected intraperitoneally (ip) with CHO cellsproducing mouse IFNγ (Mick cells). Mice receiving CHO-Mick cells willexhibit cachexia (including body weight loss) within 48 hours. Thecachectic effect is correlated with the number of Mick cells. Thus withsmall tumor cell inocula (0.8−3.0×10⁷ cells), cachexia is transient andmice will completely recover. However, with high inocula (>3.4×10⁷cells), mice continue to loose weight and will die within 7 days. It isshown that IFNγ plays an essential role in the pathogenesis of theMick-induced cachexia as monoclonals against IFNγ can reverse thewasting effect: pretreatment (day-1) with the anti-muIFNγ antibody F3inhibits cachexia.

In order to compare the effects of F3 and its scFv on the establishedcachexia model, the following experiment has been set up: mice wereinjected with 2−4×10⁷ Mick cells on day 0 and antibody preparations wereadministered ip at time points +1.5 h, +6 h, +22 h and +66 h relative tothe time of Mick cell inoculation. For scFvF3, a dose of 190 μg wasgiven each injection while for F3, 40 μg was given. Control animals wereinjected with saline at the same time points. In each group, 3 or 4 micewere used. Mice were weighed for 10 consecutive days and mortality wasscored. The results of 2 independent experiments are shown in FIG. 33.The mice treated with scFvF3 were better protected against the cachecticeffect than the control mice.

These results also indicate that scFvF3 antibody fragments do have aprotective effect of cachexia but to a lesser extent than the parentalF3 antibody. Although results were promising, it was clear that theeffect of the scFv fragment was limited either due to its fast clearanceor to lowered affinity. Optimization of the injection schedule wasneeded to obtain comparable results.

8.5. Beneficial Effects in Septic Shock in Non-Human Primates

The best documented sepsis model in non-human primates is the one inwhich baboons are given lethal infusions of E.coli. As described byCreasey et al. (1991), response to lethal E.coli challenge occurs in 3stages: an inflammatory stage marked by a fall in white blood cell count(0-2 hr) and the appearance in plasma of TNFα, IL-1β and IL-6; acoagulant stage marked by a fall in fibrinogen concentration (2-6 hr);and a hypoxic cell injury stage marked by a rise in SGPT/BUN and by agradual cardiovascular collapse, and death (6-24 hr).

Since the baboon animal model was not readily available, we areestablishing a comparable rhesus monkey model. D9D10 and derivedconstructs interacted well with rhesus IFNγ as determined in anantiviral bioassay (set up as described in example 8.2).

Septic shock can be induced by infusion either of life bacteria or ofendotoxin in sedated monkeys. After administration of differentconcentrations of the D9D10 anti-hIFNγ derivatives, several parametersare monitored including:

-   -   mortality (should be 100% in control (non-treated) group)    -   pathophysiology    -   serum concentration of cytokines such as TNFα, IL-1 and IL-6        using ELISA or bioassay (Villinger et al., 1993)    -   endotoxin profile using the limulus amoebocyte lysate assay        8.6. Beneficial Effects During Experimental Autoimmune        Encephalomyelitis in Non-human Primates        A. Pharmacokinetics of D9D10 and Derivatives in Monkey and        Effect on hIFNγ Clearance

The clearance of the antibody derivatives is of importance as moleculeswith a slow clearance have a prolonged efficacy. This implicates thatless material has to be injected which is better for the patient andwhich is cost effective, especially when a longer treatment period isadvisable. Therefore, complexes of IFNγ and D9D10 derivatives are usedin clearance studies in non-human primates as a prerequisite to guidefurther in vivo studies in these animals.

The clearance of D9D10, scFvD9D10H6⁻, D9D10 MOTAB I and D9D10 MOTAB II,is monitored after a bolus injection in healthy marmoset. SpecificELISA's are used for monitoring; no labelling of the antibody constructsis required.

Blood clearance of radiolabelled marmoset IFNγ after a bolus intravenousinjection alone or in combination with one of the antibody constructsare also performed.

B. Beneficial Effects of the D9D10 Antibody Constructs on EAE inNon-human Primates

In order to evaluate the therapeutic potential of the anti-IFNγ MabD9D10 and derivatives, we are testing this antibody in a relevantnon-human primate model for MS as the final step in our preclinicalresearch. This model is required since the antibody is notcross-reacting with IFNγ from rodents and the biological activity ofIFNγ is very species specific (huIFNγ is not active on cells other thanhuman or non-human primates (Terrell and Green, 1993)). D9D10 andderived constructs interact well with marmoset IFNγ as determined in anantiviral bioassay (set up as described in example 8.2) and usingsurface plasmon resonance (set up as described in example 1).

The EAE model is chosen as it is a generally accepted model for MultipleSclerosis. We opt for the EAE model in common marmoset (Callithrixjacchus) as it is well developed (Massacesi et al., 1995; Genain et al.,1995), it has a pathology of MR-detectable lesions which reflects thosein MS and the model shows a high incidence of EAE induction with achronic progressive/relapsing-remitting course.

Acute PK-Tox

A limited PK-Tox study required by ethical prescriptions in all researchinvolving non-human primates, is set up in order to test the toxicity ofthe substances administered either intravenously or in the lumbarcerebrospinal fluid (CSF), as the contribution of systemic and/or localIFNγ to the development of the disease is still unclear. Relatively highconcentrations of the antibody preparations, especially for the scFv,are injected intravenously as one of our goals is to reach therapeuticalconcentrations in the CNS. Although it is known that BBB leakage occursat the site of inflammation ('t Hart, personal communication), apositive concentration gradient will be beneficial.

Timing of the Study

Determination of the baseline parameters is done 1 week prior toadministration of the study drug. Animals are observed for signs oftoxicity for 30 days. During this period pharmacokinetic parameters aremonitored. Six weeks after the administration of the study drug anadditional blood sample is collected to determine whether or not theanimals mounted an immune response to any of the D9D10 constructs or torecombinant marmoset IFNγ.

Parameters

During this study the following parameters are determined:

Clinical Monitoring Daily: Food consumption Weekly: Body weights Day 14,28: Haematology Clinical chemistry Urine analysisImmunological Monitoring

Serum and CSF levels of humanized D9D10, MoTAbI and II or IFNγ aremeasured at different time points. When severe toxicity occurs in one ofthe animals, the animal are sacrificed and subjected to a detailednecropsy, in order to determine whether this toxicity is drug related.

Diffusion of D9D10 Derivatives Into the Lesions

As both systemically and locally (in the brain) produced IFNγ can have adisease promoting role in EAE, antibody derivatives must be able toneutralize both. Consequently, transudation of the D9D10 derivativesinto the lesions in brain and spinal cord is necessary for a localeffect on IFNγ. However, it is known that in MS the blood brain barrieris impaired in a subset of the active brain lesions for a limited periodof time. More specifically, BBB breakdown is reflecting the state ofinflammation (Hawkins et al., 1990).

The differential ability of the anti-IFNγ constructs to enter the brainis crucial for the choice of the component(s) which will be used forevaluation of the therapeutic efficacy of an anti-IFNγ treatment in EAE.

The entry of the constructs into the brain compartment is measured bypost-mortem magnetic resonance imaging (MRI)-scan of the brain and thespinal cord of a relapsing monkey, injected intravenously with agadolinium-diethylene-triamine pentaacetic acid (Gd-DTPA)-labelled D9D10construct 1 hour prior to sacrifice. MRI-scans are compared and arerelated to an MRI-scan taken just before death after an injection with asmall gadolinium salt that easily enters through leakages in the BBB(Gonzalez-Scarano et al., 1987; Hawkins et al., 1990; Youl et al, 1991).

These results reveal which D9D10 construct most easily enters the brainand which molecule eventually enters the active lesions where the BBB isalready restituted.

Therapeutic Treatment of Marmoset Monkeys Undergoing EAE Disease Relapse

The therapeutic effect of either systemically or locally administeredanti-IFNγ on the outcome of EAE in marmoset is evaluated. The start ofthe treatment of the monkeys is situated at the beginning of the firstrelapse of EAE, which usually occurs several months after the initialimmunization. During the experiment the following observations, analysisand measurements are carried out as of the time of relapse.

Clinical Monitoring

The severity of EAE is scored daily on an arbitrary scale modified fromMassacesi et al. (1995)

Body weight and body temperature (at time of blood sampling)

Behavioural tests for monitoring the failure of neurological functions

Magnetic resonance imaging (MRI) of the CNS

Biochemical parameters: neopterin (specifically formed in activatedmacrophages) is measured in urine

Immunological Monitoring

At several indicated time points serum is taken to monitor the bloodlevels of the antibody constructs or IFNγ and to monitor the marmosetanti-mouse or anti-IFN response.

Pathology

MRI-guided histopathology analysis has proven a powerful tool fordetailed analysis of MR-detectable lesions with histological methods.Briefly, at a chosen moment but preferably shortly after in vivoMR-images have been recorded, the monkey are euthanised. The brain andspinal cord is carefully excised and fixed in toto for 3 days in 4%buffered formaldehyde. Then a T2-weighted scan is made in axial andcoronal direction, with a slice thickness of 1 mm covering the wholebrain. For orientation of the axial slices of in vivo and in vitroimages the anterior and posterior tips of the corpus callosum are usedas internal reference points.

The excellent structural conservation and the high resolution of theMR-image make accurate three-dimensional localisation of potentiallesions possible. Regions of interest are subsequently excised andhistologically analysed for infiltrating cells (Haematoxylin-eosin),demyelisation (KLB staining of myelin lipids) and axonal structure(silver impregnation acc. to Boielschowsky).

One half of an excised brain and spinal cord is snap-frozen in liquidnitrogen. Thin cryosections are made and processed for immunohistologystaining, such as for visualisation of cytokine secreting cells(especially IFNγ) or for phenotyping of infiltrated or tissue cells.

8.7. Beneficial Effects of Anti-IFNγ Antibody Constructs in Crohn'sDisease

A. In Vitro Assay Using Patient-Derived Lymphocytes and AntigenPresenting Cells

Lymphocytes isolated from either peripheral blood or surgical specimen(lamina propria or ileum E) from patients with Crohn's disease, are usedfor assessment of cytokine profile, lymphotyping, and functionalcytotoxicity. The latter is performed by adding patient-derived antigenpresenting cells and measuring the cytokine profile. The effect ofanti-IFNγ derived antibody constructs on cytokine production ismeasured.

B Anti-IFNγ Treatment of Crohn's Disease

Patients with active Crohn's disease are infused with anti-IFNγ in adose ranging from 1 to 20 mg/kg. Responders in the study may continue toreceive repeated doses of anti-IFNγ. In all patients, clinical responsesare observed and Crohn's disease activity index (CDAI) is determined.

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1. A molecule which binds and neutralizes interferon-gamma and which ischosen from the group consisting of: a scFv comprising the humanizedvariable domain of the monoclonal antibody D9D10 a chimeric antibodycomprising the humanized variable domain of the monoclonal antibodyD9D10 a diabody comprising the humanized variable domain of themonoclonal antibody D9D10 and a multivalent antibody comprising thehumanized variable domain of the monoclonal antibody D9D10.
 2. Themolecule according to claim 1, wherein said multivalent antibody ischosen from the group consisting of triabodies and tetravalentantibodies.
 3. The molecule according to claim 2, wherein said triabodyor tetravalent antibody, comprise 3 and 4 variable domains,respectively, of several anti-interferon-gamma antibodies.
 4. Themolecule according to claim 2, wherein said triabody comprises 3identical variable domains of an anti-interferon-gamma antibody.
 5. Themolecule according to claim 2, wherein said triabody comprises 3identical D9D10 scFv's.
 6. The molecule according to claim 2, whereinsaid triabody comprises 3 identical humanized D9D10 scFv's.
 7. Themolecule according to claim 2, wherein said tetravalent antibodycomprises 4 identical domains of an anti-interferon-gamma antibody. 8.The molecule according to claim 2, wherein said tetravalent antibodycomprises 4 identical D9D10 scFv's.
 9. The molecule according to claim2, wherein said tetravalent antibody comprises 4 identical humanizedD9D10 scFv's.
 10. The molecule according to claim 2, wherein saidtetravalent antibody comprises a full-size humanized D9D10 antibody. 11.A pharmaceutical composition comprising a molecule according to claim 1or a mixture of said molecules in a pharmaceutically acceptableexcipient.
 12. A method for neutralizing interferon-gamma activity in amammal comprising administering to the mammal a pharmaceuticallyeffective amount of a molecule that binds and neutralizesinterferon-gamma, said molecule selected from the group consisting of: ascFv comprising the humanized variable domain of the monoclonal antibodyD9D10 a chimeric antibody comprising the humanized variable domain ofthe monoclonal antibody D9D10 a diabody comprising the humanizedvariable domain of the monoclonal antibody D9D10 and a multivalentantibody comprising the humanized variable domain of the monoclonalantibody D9D10.
 13. The method according to claim 12 wherein thepharmaceutically effective amount of the molecule that binds andneutralizes interferon-gamma is administered to prevent or treatingseptic shock, cachexia, autoimmune disease(s), and/or skin disorder(s).14. The method of claim 13 wherin the autoimmune disease is multiplesclerosis, Crohn's disease, or rheumatoid arthritis.
 15. The method ofclaim 13 wherein the skin disorder is bullous, inflammatory, orneoplastic dermatosis.
 16. The method of claim 12 wherein saidmultivalent antibody is chosen from the group consisting of triabodiesand tetravalent antibodies.
 17. The method according to claim 12 whereinsaid triabody or tetravalent antibody, comprise 3 and 4 variabledomains, respectively, of several anti-interferon-gamma antibodies. 18.The method according to claim 12 wherein said triabody comprises 3identical variable domains of an anti-interferon-gamma antibody.
 19. Themethod according to claim 12 wherein said triabody comprises 3 identicalD9D10 scFv's.
 20. The method according to claim 12 wherein said triabodycomprises 3 identical humanized D9D10 scFv's.
 21. The method accordingto claim 12 wherein said tetravalent antibody comprises 4 identicaldomains of an anti-interferon-gamma antibody.
 22. The method accordingto claim 12 wherein said tetravalent antibody comprises 4 identicalD9D10 scFv's.
 23. The method according to claim 12 wherein saidtetravalent antibody comprises 4 identical humanized D9D10 scFv's. 24.The method according to claim 12 wherein said tetravalent antibodycomprises a full-size humanized D9D10 antibody.
 25. A method fordetermining interferon gamma levels in a sample comprising: a)contacting the biological sample to be analysed for the presence ofinterferon-gamma with a molecule according to claim 1; b) detecting theimmunological complex formed between interferon-gamma and said molecule.